WO2018175854A1 - Procédés et compositions se rapportant aux adjuvants - Google Patents

Procédés et compositions se rapportant aux adjuvants Download PDF

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WO2018175854A1
WO2018175854A1 PCT/US2018/023970 US2018023970W WO2018175854A1 WO 2018175854 A1 WO2018175854 A1 WO 2018175854A1 US 2018023970 W US2018023970 W US 2018023970W WO 2018175854 A1 WO2018175854 A1 WO 2018175854A1
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adjuvant
subject
administration
kit
composition
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PCT/US2018/023970
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English (en)
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Ofer Levy
David Dowling
Helene Bazin-Lee
David Burkhart
Jay Evans
Alyson SMITH
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The Children's Medical Center Corporation
University Of Montana
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Priority to US16/495,901 priority Critical patent/US11464854B2/en
Publication of WO2018175854A1 publication Critical patent/WO2018175854A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles

Definitions

  • HHSN272201400052C awarded by the Department of Health and Human Services. The government has certain rights in the invention.
  • the technology described herein relates to adjuvants, e.g, for use in immunization.
  • Vaccines typically rely upon adjuvants to stimulate the immune system and generate an effective response to the vaccine.
  • Existing adjuvants while effective in adults, often give poor performance or are even counterproductive in infants and newborns. In order to successfully immunize infants and newborns, and reduce the number of vaccine doses such patients receive, effective adjuvants are necessary.
  • agonists of TLR7 and/or TLR8 provide surprisingly effective adjuvant activity in newborns, improving the efficacy of vaccination and lowering the number of doses required. Morever, such adjuvants permit effective vaccination at or within days of birth of the subject, providing earlier protection, reducing the number of vaccine doses required to achieve protection, and making vaccination more plausible for many at-risk populations. Such adjuvants also enhance vaccine responses during infancy, reducing the number of vaccine doses required to achieve protection.
  • a method of immunizing a subject comprising administering to the subject i) an adjuvant comprising an agonist of TLR7 and/or TLR8; and ii) at least one antigen; wherein the adjuvant and the at least one antigen are not conjugated to each other.
  • the adjuvant is lipidated. In some embodiments of any of the aspects, the adjuvant is 3M-052.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is selected from the group consisting of: a single sstranded (ss) RNA; an imidazoquinoline; a thiazoquinoline; an oxoadinine; and a benzazepine.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound having the structure of Formula IX:
  • n is from 0 to 20
  • R is R is selcted from H, Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl- 6alkyl, C3-6cycloalkylCl-6alkylamino, C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl- 6alkyl, Cl-6alkoxyCl-6alkylamino and Cl-6alkoxyCl-6alkoxy; wherein the Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl-6alkyl, C3-6cycloalkylCl-6alkylamino, 20 C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl-6alkyl, Cl-6alkoxyCl-6alkylamino or Cl- 6alkoxyCl-6alkoxy is branched or unbranched and optionally terminally substituted with a hydroxyl, amino, thio, hydrazino
  • X is a phospholipid, lipid, lipidation, and/or PEG moiety.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound having the structure of Formula X:
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound having the structure of Formula XI:
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound selected from the group consisting of: 3M-052; CRX-648; CRX- 649; CRX-664; CRX-672; CRX-677; and CRX-748. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises CRX-649.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 further comprises a lipid moiety.
  • the adjuvant further comprises a phosphorylation or phospholipid moiety.
  • the moiety is located at the ethanol group of 3M-052; CRX- 648; CRX-649; CRX-664; CRX-672; CRX-677; or CRX-748.
  • the moiety is located at an N position corresponding to the Nl of Formula X.
  • the moiety is conjugated to the adjuvant via a PEG linker.
  • the PEG linker comprises from 3 to 9 repeats of PEG. In some embodiments of any of the aspects, the PEG linker comprises 3 repeats of PEG.
  • the administration of the adjuvant and antigen causes a greater immune response, increased rate of an immune response and/or greater protection than the same dose of the antigen administered without the adjuvant. In some embodiments of any of the aspects, the administration of the adjuvant and antigen provides protection at a lower dose or with fewer doses than the antigen administered without the adjuvant.
  • the at least one antigen is comprised by an attenuated vaccine.
  • the antigen is comprised by a subunit vaccine or recombinant subunit vaccine.
  • the antigen is comprised by a conjugate vaccine.
  • the antigen is a polysaccharide.
  • the antigen is bound to or adsorbed to alum.
  • the antigen is comprised by a vaccine selected from the group consisting of a pneumococcal vaccine; a hepatitis B (HBV) vaccine; an acellular pertussis (aP) vaccine; a diphtheria tetanus acellular pertussis (DTaP) vaccine; a hepatitis A (HAV) vaccine; and a meningococcal (MV) vaccine.
  • a vaccine is pneumococcal conjugate vaccine (PCV)13.
  • the vaccine is alum-adjuvanted.
  • the method further comprises administering a second adjuvant.
  • the second adjuvant is alum.
  • the subject is a human infant at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than 28 days of age at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than 4 days of age at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than 2 days of age at the time of
  • the subject is a human of less than 24 hours of age at the time of administration. In some embodiments of any of the aspects, the administration occurs at birth.
  • the adjuvant is administered at a dose of from about 0.01 mg per kilogram of a subject's body mass to about 1.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant is administered at a dose of about 0.1 mg per kilogram of the subject's body mass.
  • the adjuvant is administered
  • the method further comprises at least a second administration of the adjuvant and antigen.
  • the first administration occurs when the subject is less than 1 day of age. In some embodiments of any of the aspects, the first administration occurs at the birth of the subject. In some embodiments of any of the aspects, the first administration occurs when the subject is less than 28 days of age. In some embodiments of any of the aspects, the first and/or second
  • the first and/or second administration occur when the subject is less than 6 months of age. In some embodiments of any of the aspects, the first and/or second administration occur when the subject is less than 28 days of age. In some embodiments of any of the aspects, the first and/or second administration occur when the subject is from 28 days to 6 months of age. In some embodiments of any of the aspects, the second administration occurs within 28 days of the first administration.
  • the adjuvant and the antigen are administered in the same formulation. In some embodiments of any of the aspects, the adjuvant and the antigen are administered in different formulations and/or at different times.
  • the antigen is administered only once. In some embodiments of any of the aspects, the antigen and adjuvant are administered only once. In some embodiments of any of the aspects, the antigen is administered no more than twice. In some embodiments of any of the aspects, the antigen and adjuvant are administered no more than twice each. In some embodiments of any of the aspects, the antigen is administered no more than three times. In some embodiments of any of the aspects, the antigen and adjuvant are administered no more than three times each.
  • the method comprising administering to the human an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • the immune response is T helper 1- cytokine production.
  • the immune response is an increase in the level of Thl CRM-197-specific neonatal CD4+ cells.
  • the adjuvant is selected from the group consisting of a single sstranded (ss) RNA; an imidazoquinoline; a thiazoquinoline; and a benzazepine.
  • the adjuvant is lipidated.
  • the adjuvant is 3M-052.
  • the method further comprises administering a second adjuvant.
  • the second adjuvant is alum.
  • the subject is a human infant at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than 28 days of age at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than 4 days of age at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than 2 days of age at the time of
  • the subject is a human of less than 24 hours of age at the time of administration. In some embodiments of any of the aspects, the administration occurs at birth.
  • the adjuvant is administered at a dose of from about 0.01 mg per kilogram of a subject's body mass to about 1.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant is administered at a dose of about 0.1 mg per kilogram of the subject's body mass.
  • the adjuvant is administered
  • the method further comprises at least a second administration of the adjuvant and antigen and/or the subject is administered at least a second administration of the adjuvant and antigen, and/or the composition or kit further comprises a second dose of the adjuvant and antigen.
  • the first administration occurs when the subject is less than 1 day of age. In some embodiments of any of the aspects, the first administration occurs at the birth of the subject. In some embodiments of any of the aspects, the first administration occurs when the subject is less than 28 days of age. In some embodiments of any of the aspects, the first and/or second administration occur when the subject is less than 6 months of age.
  • the first and/or second administration occur when the subject is less than 28 days of age. In some embodiments of any of the aspects, the first and/or second administration occur when the subject is from 28 days to 6 months of age. In some embodiments of any of the aspects, the second administration occurs within 28 days of the first administration.
  • compositions for use in immunizing a subject or stimulating an immune response in a subject comprising an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • compositions comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 are provided.
  • the composition further comprises at least one antigen, wherein the adjuvant and the at least one antigen are not conjugated to each other.
  • composition or kit comprising a first formulation comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 and a second formulation comprising at least one antigen, wherein the formulations are for use in immunizing a subject or stimulating an immune response in a subject.
  • a composition or kit comprising a first formulation comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 and a second formulation comprising at least one antigen.
  • kits comprising an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • the composition or kit can further comprise at least one antigen.
  • the at least one antigen is comprised by an attenuated vaccine.
  • the antigen is comprised by a subunit vaccine or recombinant subunit vaccine.
  • the antigen is comprised by a conjugate vaccine.
  • the antigen is a polysaccharide.
  • the antigen is bound to or adsorbed to alum.
  • the antigen is comprised by a vaccine selected from the group consisting of a pneumococcal vaccine; a hepatitis B (HBV) vaccine; an acellular pertussis (aP) vaccine; a diphtheria tetanus acellular pertussis (DTaP) vaccine; a hepatitis A (HAV) vaccine; and a meningococcal (MV) vaccine.
  • a vaccine is pneumococcal conjugate vaccine (PCV)13.
  • the vaccine is alum-adjuvanted.
  • composition or kit further comprises a second adjuvant.
  • the second adjuvant is alum.
  • the adjuvant is formulated at a dose of from about 0.01 mg per kilogram of a subject's body mass to about 1.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant is formulated at a dose of about 0.1 mg per kilogram of the subject's body mass.
  • Figs. 1 A- ID demonstrate that 3M-052 is a lipidated, locally-acting TLR7/8 Agonist that, unlike R848, does not result in robust systemic levels and systemic cytokine production.
  • Figs. 2A-2D demonsrate that 3M-052 synergistically enhances Type 1 immunity from newborn leukocytes when combined with pneumococcal conjugate vaccine in vitro.
  • Fig. 3 A depicts the rhesus macaque study groups and their enrollment/immunization timeline. Neonatal and infant rhesus macaques were immunized at day of life (DOL)-O, 28, and 56 (the three immunization time-points are indicated by boxes) with either PC VI 3 alone or PC VI 3 co-administered with 3M-052 (a lipidated TLR7/8A).
  • Horizontal broken line indicates the WHO- recommended reference Ab concentration of IgG used as a correlate of protection levels in humans (0.35 ⁇ g/ml). Numbers refer to p values approaching significant for that group. For comparisons between overall groups (e.g., PC VI 3 vs. (PC VI 3 + 3M-052)), statistical significance denoted as +p ⁇ 0.05, ++p ⁇ 0.01 or NS (not significant).
  • Figs 5A-5D demonstrate that (PC VI 3 + 3M-052) activates both Thl7 and Thl CRM- 197-specific CD4+ cells.
  • Figs. 5A-5C depict the percentage of IL-4-, IL-17-, and IFNy- producing CRM197-specific CD4+ T cells post-ex vivo recall assays with CRM197-pulsed autologous rhesus DCs.
  • 5D depicts pie charts representing scale and frequencies of cytokine producing CRM197-specific CD4+ T cells, indicating that neonatal PCV13 alone treatment enhances CRM197-specific Thl7-responses, while (PCV13 + 3M-052) enhances and accelerates a mixed Thl/Thl7-response.
  • Figs. 6A-6C demonstrate that 3M-052 enhances and accelerates activation of early life PnPS-specific B cells.
  • Fig. 6C demonstrates that co-administration of 3M-052 with PCV13 to newborn rhesus macaques dramatically accelerated the transition of anti-PnPS B-cells from naive to memory phenotype.
  • Figs. 7A-7B demonstrate Cytokine and IFN-inducible gene expression following free or lipidated TLR7/8 imidazoquinoline subcutaneous injection.
  • Mouse mRNA expression is depicted in (Fig. 7A) draining lymph nodes (brachial and axillary), and (Fig. 7B) spleen post a single subcutaneous injection of 3M-052 or R848 formulated (both 1 mg/kg, (20 ⁇ g/mouse)) in oil-in-water emulsion (O/W) (vehicle) to the scruff of the neck.
  • Figs. 8A-8B demonstrate that addition of 3M-052 to PCV13 enhances TNF and IFN responses in newborn cord blood.
  • Human neonatal and adult blood was cultured for 6 hours with sterile buffer control (RPMI, not shown), PCV13 (1 :5.7 - 57,000 v/v), 3M-052 or R848 (both 0.01, 0.1, 1, 10, 100 ⁇ ), or (PCV13+3M-052).
  • Fig. 9 demonstrates that human newborn whole blood cytokine responses to 3M-052, PCV13, and (PC VI 3 + 3M-052).
  • overall groups e.g., PCV13 vs.
  • Fig. 10 demonstrates that human adult whole blood cytokine responses to 3M-052, PCV13 and (PC VI 3 + 3M-052).
  • overall groups e.g., PCV13 vs.
  • Figs. 1 1 A-l IB demonstrate that 3M-052 enhances antigen-specific IgG levels while also skewing the response towards Thl (IgG2a induction).
  • Figs. 12A-12C demonstrate that addition of 3M-052 augments Thl -responses to alum adjuvanted influenza hemagglutinin antigen. Addition of 3M-052 to Alumadjuvanted HA antigen markedly enhances IgG2a Ab production.
  • Balb/c mice were immunized by subcutaneous injection (scruff of neck) with a 10 ⁇ g dose of influenza hemagglutinin (HA) alone or in combination with Alum or 0.1 mg/kg 3M-052 three times (prime, boost, boost) 14 days apart.
  • Fig. 13 demonstrates that Addition of a TLR7/8 agonist accelerates serotype-specific antibody responses to PCV13 in a dose dependent manner.
  • Fig. 14 demonstrates that TLR7/8 adjuvantation markedly accelerates and enhances serotype-specific pneumococcal opsonophagocytic killing capacity in neonatal serum.
  • Neonatal and infant rhesus macaques were immunized at DOLO, 28, and 56 with either PCV13 alone or (PC VI 3 + 3M-052).
  • Peripheral blood was collected at the indicated time-points to obtain serum for measurement of IgG concentrations and opsonization indicies (OIs) as described in Example 1.
  • Geometric mean titers of serotype-specific opsonophagocytic killing activity from n 3 rhesus macaques per treatment group are shown. Samples with undetectable OIs were assigned an 01 of 12. Results represent means ⁇ SEM.
  • Figs. 15A-15B demonstrate that TLR7/8 adjuvantation dramatically accelerates and enhances serotype-specific pneumococcal opsonophagocytic killing capacity in neonatal serum.
  • Neonatal and infant rhesus macaques were immunized at DOLO, 28, and 56 days with either PCV13 alone or PC VI 3 co-administered with 3M-052.
  • Peripheral blood was collected at the indicated time-points to obtain serum.
  • OIs opsonization indices
  • Fig. 16 demonstrates day 28 opsonophagocytic killing activity corresponds with accelerated serotype-specific antibody responses to TLR7/8 agonistadjuvanted pneumococcal conjugate vaccine.
  • Neonatal and infant rhesus macaques were immunized at DOLO with either PCV13 alone or (PCV13 + 3M-052).
  • Peripheral blood was collected at the indicated time-points to obtain serum for measurement of IgG concentrations and opsonization indicies (OIs) as described in Example 1.
  • Fig. 17 demonstrates that TLR7/8 agonist-adjuvantation of PCV13 enhances Day 56 opsonophagocytic killing activity.
  • Neonatal and infant rhesus macaques were immunized at DOLO and 28 with either PCV13 alone or (PC VI 3 + 3M-052).
  • Peripheral blood was collected at the indicated time-points to obtain serum for measurement of IgG concentrations and
  • Fig. 18 demonstrates that TLR7/8 agonist-adjuvantation of PCV13 enhances Day 120 opsonophagocytic killing activity. Neonatal and infant rhesus macaques were immunized at DOL0, 28, and 56 with either PC VI 3 alone or (PCV13 + 3M-052).
  • Figs. 19A-19C demonstrate that weight and body temperature of immunized neonatal and infant rhesus macaques.
  • Fig. 19 A Weight, a sensitive indicator of neonatal wellbeing, was measured regularly to DOL150 and are depicted as normalized values relative to birth weight (100%) for each treatment group. Dotted lines indicate normal age-matched norms with standard deviations.
  • Fig. 19B Body temperature was measured by rectal thermometer at regular intervals up to DOL150.
  • Fig. 19C Body temperatures pre- and post- each immunization at DOL0, 28, and 56. For comparison at individual time-points, the unpaired Mann-Whitney test was applied, with statistical significance denoted as *p ⁇ 0.05. Results represent means ⁇ SEM of 3-5 animal's per group.
  • Figs. 20A-20B demonstrate that Intramuscular injection of 3M-052 induces injection site erythema post-second immunization. Neonatal and infant rhesus macaques were immunized at DOLO, 28, and 56 with either PCV13 alone or (PC VI 3 + 3M-052).
  • Figs. 21 A-21D demonstrate that 3M-052 administration with or without PCV13 does not induce systemic cytokines in neonatal/infant rhesus macaques.
  • Figs. 22A-22B demonstrate experimental approach used for mononuclear cell sorting and ex vivo assessment of vaccine-specific B and T cells in infant rhesus macaques.
  • Fig. 22A Sorted leukocytes were incubated as depicted. B cell subsets (left) were non-specifically stimulated with R848/IL-2/IFN ⁇ to induce differentiation to Ab-secreting plasma cells. Plasma cells were subsequently plated on ELISpot plates for detection of pneumococcal polysaccharide (PnPS)-specific B cells. Monocytes were differentiated to monocyte-derived dendritic cells (MoDCs) by the addition of GM-CSF and IL-4.
  • PnPS pneumococcal polysaccharide
  • Fig. 22B Frozen PBMCs were thawed and stained with CD20- Pacific Blue, CD27-PE.Cy7, CD14-PE, CD4-FITC, and CD8-APC.Cy7. Cells were subsequently sorted according to the gating strategy depicted on a FACSAria II cytometer.
  • Figs. 23 A-23D demonstrate that 3M-052 accelerated and enhanced the magnitude of neonatal and infant anti-PnPS antibody (IgG) responses and may enhance antibody avidity.
  • Fig. 24 demonstrates that co-administration of 3M-052 with PCV13 increased infiltration of CD68+ cells at the vaccine injection site.
  • Immunization with (PC VI 3 + 3M-052) accelerates injection site infiltration by monocytes/macrophages.
  • 2 mm cube muscle biopsies were obtained from the injection site (quadriceps muscle) prior to and 48 hours after each immunization (obtained in an alternating pattern (e.g. DOL0 left leg, DOL2 right leg)).
  • Frequencies of CD68+ cells in muscle were determined by immunofluorescence. For comparison at individual time-points, the unpaired Mann-Whitney test was applied, with statistical significance denoted as *p ⁇ 0.05, **p ⁇ 0.01, or NS (not significant). Data are representative of two animals per treatment group.
  • Figs. 25A-25C depict an overview of the core-aqueous imidazoquinoline and oxoadenine scaffolds.
  • Fig. 25 depicts a table of naming convention, chemical class and TLR selectively of each core scaffolds;
  • Fig. 25B depicts structures of core imidazoquinolines;
  • Fig. 25C depicts structures of core oxoadenines investigated in this study.
  • Figs. 26A-26C demonsrate that CRX-649 has the greatest potency for both hTLR7 and hTLR8.
  • Six TLR agonists were compared. HEK-293 cells transfected with human.
  • Fig. 26C) TLR7 and (Fig. 26B) TLR8 and an F-kB-driven reporter SEAP gene were stimulated for 18-24 h with TLR agonists.
  • the y-axis shows the level of SEAP activity as a fold change over unstimulated cells.
  • the x-axis shows the concentration of each compound in ⁇ .
  • Each data point represents the mean of triplicate culture wells, and representative of three separate experiments.
  • Fig. 26C demonstrates that amongst the IMQ and OA compounds evaluated in a HEK293 assay, CRX-is the most potent for both TLR7 and TLR8, with a preference for TLR8.
  • Figs. 27A-27D demonstrate that imidazoquinoline CRX-649 demonstrates age- specific potency, effectiveness and IFN ⁇ polarization in newborn cord blood.
  • Figs. 27A-27B depict experiments with human neonatal blood cultured in vitro for 6 hours with buffer control (RPMI) or with increasing concentrations of various CRX adjuvants.
  • Figs. 27C-27D depicts experiments with newborn versus adult blood cultured in vitro for 6 hours with CRX-649.
  • Figs. 28A-28F demonstrate that imidazoquinoline CRX-649 demonstrates newborn- specific cytokine and chemokine potency and polarization.
  • Figs. 28A, 28C cytokine and interferon production
  • Figs. 28B, 28D chemokine and growth factor production.
  • FIG. 28E depicts flow cytometry analysis of human adult PBMCs stimulated with CRX-649 for 24 h.
  • CD123, HLA-D, CD80 and CD86 all show increases as compared to unstimulated cells.
  • Fig. 28F depicts newborn CBMCs stimulated with CRX-649, in the presence of the polyclonal T cell activator aCD3 for 96 hours.
  • IFN ⁇ levels were measured in cell-free supernatants by ELISA. Results are shown as the median, the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) of 5 independent experiments.
  • N 4. For comparison at individual time points, the unpaired Mann-Whitney test was applied, *p ⁇ 0.05.
  • Figs. 29A-29E demonstrate that lipidation of the basic FMQ scaffold changes the immunostimulatory properties in adult PBMCs.
  • Fig. 29A depicts the basic imidazoquinoline (FMQ) pharmacophore of TLR7/8 agonist;
  • Fig. 29B depicts optimized core CRX-649;
  • Fig. 29C depicts the lead TLR7/8 agonist CRX-727.
  • PEG Poly Ethylene Glycol
  • Fig. 30 depicts a table of lipidated TLR7/8 adjuvant CRX-727 rapidly and fully adsorbs to the alum/antigen while the core compound CRX-649 does not.
  • CRX-727 fully adsorbed (-96 - 100 %) to the alum/antigen within 1 hr, with or without excess alum (top panels).
  • the core CRX-649 compound was only able to adsorb to the antigen ⁇ 4 - 7 % within 1-2 hr (bottom panels), with peak area intensity levels similar to the unmixed controls, mAU: milli-absorbance units.
  • the phosphorylated CRX-649 derivative, CRX-650 was also included as a control.
  • Figs. 31 A-31C demonstrate that antibody production in response to Infanrix with or without different formulations of CRX-727.
  • Balb/c mice were immunized twice, 14 days apart (Fig. 31 A) with Infanrix (l/lOO 111 of the human dose) ⁇ CRX649 or CRX-727 at 0.1 ⁇ g, 1 ⁇ g or 10 ⁇ g per mouse in different formulations (aqueous or pre-adsorbed to alum).
  • Serum was harvested 14 days following prime (Fig. 21B) or boost (Fig. 31C) and anti-FHA serum antibody IgGl and IgG2a titers were measured by ELISA.
  • Fig. 32 demonstrates higher percentages of IFNy-producing CD4 + T cells after vaccination of DTaP with CRX-727 or alum adsorbed CRX-727.
  • Balb/c mice were immunized twice, 14 days apart with Infanrix (l/100 th of the human dose) ⁇ CRX649 or CRX-727 at 0.1 ⁇ g, 1 ⁇ g or 10 ⁇ g per mouse in different formulations.
  • Cell-mediated immune response in spleens from 3 mice per group were harvested 5 days post-secondary immunization and restimulated with purified pertussis antigen followed by intracellular cytokine staining and analysis via flow cytometry. Data is represented as percentage IFNy-positive CD4 + T cells.
  • Figs. 33A-33E demonstrate that antibody production in neonatal mice immunized with Infanrix combined with various formulations and doses of CRX-727.
  • Fig. 33 A depicts the results when 7 day old C57BL/6, 4-6 per group, were vaccinated (prime/ boost) with Infanrix (1/100 & of the human dose), ⁇ CRX649 or CRX-727 at 0.1 ⁇ g, 1 ⁇ g or 10 ⁇ g per mouse in different formulations (aqueous, liposome or alum adsorbed "Alum Abs").
  • Serum was harvested 14 days following boost (14dp2) (DOL 28) and anti-FHA serum total IgG titers (Fig.
  • FIG. 33B depicts the fold change analysis for antibody production with a 0.1 ⁇ dose of CRX-727 + DTaP, as compared DTaP alone (N; newborn, A; adult).
  • Statistical comparison employed test by one way ANOVA; **** ⁇ 0.0001, with comparison to Infanrix alone.
  • Fig. 34 depicts graphs demonstrating TNF and IFNa induced by PEGylated imidazoquinolines in human adult PBMCs.
  • Infection is the most common cause of mortality in early life and immunization is the most promising biomedical intervention to reduce this burden.
  • newborns fail to optimally respond to most vaccines.
  • Patient responses to vaccines are routinely enhanced by administering an adjuvant as a component of the vaccine formulation, but currently utilized adjuvants do not provide sufficient responses in newborns.
  • This improved vaccine response permits the use of fewer doses and/or lower doses of vaccine while also permitting effective vaccination at or immediately after birth, which is a critical concern in less-developed areas where regular post-delivery medical care is not common.
  • TLR7/8 adjuvant provides the desired enhancements of the immune response, thereby providing methods and compositions that permit successful vaccination of newborns.
  • the adjuvants described herein are not complexed with the vaccine antigen itself, allowing the adjuvant to be administered separately or readily mixed with currently used vaccine formulations to enhance the immune response. This is a distinct advantage over many current adjuvants, which are bound to or complexed with the relevant antigen.
  • described herein is a method of method of immunizing a subject, the method comprising administering to the subject i) an adjuvant comprising an agonist of TLR7 and/or TLR8; and ii) at least one antigen; wherein the adjuvant and the at least one antigen are not conjugated to each other.
  • described herein is a method of stimulating an immune response of a subject, the method comprising administering to the human an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • administration of an adjuvant comprising an agonist of TLR7 and/or TLR8, either with or without an antigen can result in, e.g., a greater immune response, increased rate of an immune response and/or greater protection than in the absence of the adjuvant.
  • administration of an adjuvant comprising an agonist of TLR7 and/or TLR8 and an antigen as described herein can provide protection at a lower dose or with fewer doses than the antigen administered without the adjuvant.
  • adjuvant refers to any substance than when used in combination with a specific antigen that produces a more robust immune response than the antigen alone.
  • an adjuvant acts generally to accelerate, prolong, or enhance the quality of specific immune responses to the vaccine antigen(s).
  • TLR7 or “Toll-like receptor 7” refers to a transmembrane protein of the toll-like receptor family that recognizes ssRNA, parti culary GU-rich ssRNA of viral origin. Sequences for TLR7 are known for a number of species, e.g., human TLR7 (NCBI Gene ID: 51284) mRNA sequences (NM_016562.3) and polypeptide sequences (NP_057646.1).
  • TLR8 or “Toll-like receptor 8” refers to a transmembrane protein of the toll-like receptor family that recognizes ssRNA, parti culary GU-rich or G-rich ssRNA of viral origin. Sequences for TLR8 are known for a number of species, e.g., human TLR8 (NCBI Gene ID: 51311) mRNA sequences (NM_016610.3 and NM_138636.5) and polypeptide sequences (NP_057694.2 and NP_619542.1).
  • the term "agonist" refers to an agent which increases the expression and/or activity of the target by at least 10% or more, e.g. by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000 % or more.
  • the efficacy of an agonist of, for example, TLR7 and/or TLR8, e.g. its ability to increase the level and/or activity of TLR7 and/or TLR8 can be determined, e.g. by measuring the level of an expression product of TLR7 and/or TLR8 and/or the activity of TLR7 and/or TLR8.
  • RT-PCR with primers can be used to determine the level of RNA
  • Western blotting with an antibody can be used to determine the level of a polypeptide.
  • suitable primers are provided in the Examples herein and antibodies to TLR7 and/or TLR8 are commercially available, e.g., Cat. No. ab45371 and ab24185 from Abeam (Cambridge, MA).
  • Assays for measuring the activity of TLR7 and/or TLR8, e.g. the increases in cytokine production, cell proliferation, and cell survival in response to ssRNA detection are known in the art.
  • an adjuvant described herein can comprise an agonist of TLR7 and/or TLR8. In some embodiemnts of any of the aspects, an adjuvant described herein can consist essentially of an agonist of TLR7 and/or TLR8. In some embodiemnts of any of the aspects, an adjuvant described herein can consist of an agonist of TLR7 and/or TLR8.
  • an agonist of TLR7 and/or TLR8 can be an agonist of TLR7 but not TLR8. In some embodiments of any of the aspects, an agonist of TLR7 and/or TLR8 can be a specific agonist of TLR7. In some embodiments of any of the aspects, an agonist of TLR7 and/or TLR8 can be an agonist of TLR8 but not TLR7. In some embodiments of any of the aspects, an agonist of TLR7 and/or TLR8 can be a specific agonist of TLR8. In some embodiments of any of the aspects, an agonist of TLR7 and/or TLR8 can be a specific agonist of TLR7 and TLR8. In some embodiments of any of the aspects, an agonist of TLR7 and/or TLR8 can be an agonist of TLR7 and TLR8.
  • TLR7 and/or TLR8 are known in the art and can include, by way of non-limiting example, a single sstranded (ss) RNA; a ssRNA derived from a viral pathogen (e.g., HIV, HCV, influenza, Sendai, and Coxsackie viruses); an imidazoquinoline (e.g., R-848 (Formula III below), 3M-002 (CL075; Formula I below), 3M-013, 3M003 (4-amino-2-(ethoxymethyl)- ⁇ , ⁇ -dimethyl- 6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-l-ethanol.hydrate), R837 (Imiquimiod, 4-amino- 2ethoxymethyl-a,a-dimethyl-lH-imidazo[4,5-c]quinolines-l-ethanol, l-(2-methylpropyl)-lH- imidazo[4,5-c]quinol
  • TLR7 guanosine analogues
  • TOG 7-thia-8-oxo-G
  • Ioxoribine 7-allyl-8-oxo-G
  • TLR7 agonists can also include (1) guanosine analogues, such as 7-deazaguanosine and related compounds, including those described in Townsend, (1976) Heterocyclic Chem, 13, 1363, and Seela, et al, (1981) Chem. Ber., 114(10), 3395-3402; 7-allyl, 8-oxo-guanosine
  • imidazoquinolines including l-(4-amino-2-ethoxymethyl-imidazo[4,5- c]quinolin-l-yl)-2-methyl-propan-2-ol (imiquimod), as described in WO 94/17043; 1-isobutyl- lH-imidazo[4,5-c]quinolin-4-ylamine (resiquimod) as described in WO 94/17043 and US 2003/0195209, US 2003/0186949, US 2003/0176458, US 2003/0162806, 2003/0100764, US 2003/0065005 and US 2002/0173655); U.S. Pat. No.
  • US 2008/0171712 describes a novel class of stabilized immune modulatory RNA (SFMRA) compounds which bind to TLR7 and TLR8.
  • SFMRA compounds that specifically activate TLR7 especially the compounds having a structure as set out in Formulas I-IV in Table 2, and specific compounds listed in Table 4, are described in US 2010/0215642 (Idera).
  • TLR7 agonists including lipid-linked TLR7 agonists, are described in US 2010/0210598 (Regents of the University of California, San Diego). TLR7 agonists, including orally-available-linked TLR7 agonists and TLR7agonist prodrugs, are described in US 2010/0210598 (Regents of the University of California, San Diego). TLR7 agonists, including orally-available-linked TLR7 agonists and TLR7agonist prodrugs, are described in US
  • Non-selective TLR7 agonists are described in US 2009/0324551 (The Regents of The University of California).
  • Immunostimulatory polymers that contain sequence-dependent immunostimulatory RNA motifs and methods for their use are described in US 2010/0272785.
  • the sequence-dependent immunostimulatory RNA motifs and the polymers incorporating such motifs are selective inducers of TLR7and the TLR7-associated cytokine IFN-a (Coley Pharmaceutical).
  • US 2010/0029585 and WO 2010/014913 describe formulations of benzo[b]azepine compounds that are TLR7 and/or TLR8 agonists.
  • TLR8 agonists that may be suitable in the context of the present invention include VTX-1463 and VTX-2337 (VentiRx Pharmaceuticals), both of which have successfully completed phase I clinical trials.
  • a review article concerning TLR8 agonists is Philbin & Levy (2007) "Immunostimulatory activity of Toll-like receptor ⁇ agonists towards human leucocytes: basic mechanisms and translational opportunities”. Biochemical Society Transactions 35(6): 1485-90.
  • Each of the foregoing references is incorporated by reference herein in its entirety.
  • the adjuvant comprising a TLR7 and/or TLR8 agonist can comprise a compound having the structure of Formula IX, wherein n is from 0 to 20, R is R is selcted from H, Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl- 6alkyl, C3-6cycloalkylCl-6alkylamino, C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl-6alkyl, Cl- 6alkoxyCl-6alkylamino and Cl-6alkoxyCl-6alkoxy; wherein the Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl-6alkyl, C3-6cycloalkylCl-6alkylamino, 20 C3-6cycloalkylCl- 6alkoxy, Cl-6alkoxyCl-6alkyl, Cl-6al
  • the adjuvant comprising a TLR7 and/or TLR8 agonist can comprise a compound having the structure of Formula X.
  • the adjuvant comprising a TLR7 and/or TLR8 agonist can comprise a compound having the structure of Formula XI.
  • the adjuvant comprising a TLR7 and/or TLR8 agonist can comprise a compound having the structure of Formula XII, wherein n is from 1 to 15 and R2 is a lipid group.
  • the R 2 group of Formula XII is:
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound selected from the group consisting of: 3M-052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; and CRX-748.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 consists essentially of a compound selected from the group consisting of: 3M-052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; and CRX-748.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 consists of a compound selected from the group consisting of: 3M-052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; and CRX-748.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises CRX-649 (Formula X).
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 consists essentially of CRX- 649.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 consists of CRX-649.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises CRX-727 (Formula XI). In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 consists essentially of CRX- 727. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 consists of CRX-727.
  • the adjuvant can comprise one agonist of TLR7 and/or TLR8. In some embodiments of any of the aspects, the adjuvant can comprise two or more agonists of TLR7 and/or TLR8, e.g., two different agonists, three different agonists, or more agonists.
  • the adjuvant can comprise a phosphate group and/or a phospholipidation moiety, e.g., a phospholipid can be covalently bounded to the adjuvant (e.g., to the embodiments of adjuvants described above herein).
  • Lipidation can prevent or inhibit migration of the adjuvant, increasing local activity.
  • the adjuvant can be lipidated and/or comprise a lipid moiety, e.g., a lipid can be covalently bonded to the adjuvant (e.g., to the embodiments of adjuvants described above herein).
  • the adjuvant comprises a lipid covalently bound to the agonist of TLR7 and/or TLR8. Examples of lipids for used in the lipidated adjuvants described herein can include, but are not limited to C18 lipid moieties.
  • the lipid and/or phospholipid moiety can be located at (e.g., conjugated to) the ethanol group of 3M-052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; or CRX-748 or an ethanol group corresponding to the ethanol group of 3M-052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; or CRX- 748.
  • the lipid and/or phospholipid moiety can be located at (e.g., conjugated to) the Nl position of Formula X or an N corresponding to the Nl position of Formula X.
  • the adjuvant and the lipid can be covalently conjugated with each other using a reactive functional group present in their respective structures.
  • the term "reactive functional group” refers to a functional group that is capable of reacting with another functional group. Exemplary reactive functional groups include, but are not limited to, hydroxyls, amines, thiols, thials, sulfinos, carboxylic acids, acyl chlorides, amides, and the like.
  • the reactive functional group on the lipid and the adjuvant can be the same or different.
  • the reactive group on the lipid is a carboxylic acid, a carboxylic acid derivative such as acid chloride or an ester, a hydroxyl, an amine or a thiol.
  • the reactive group on the adjuvant is an amine, a hydroxyl, a thiol, or a carboxylic acid.
  • the amine can be acyclic, cyclic, aromatic amine, or heterocyclic amine.
  • Some preferred amines in some aspects of the invention include, but are not limited to imidazole, aniline, indole, pyridine, piperidine, pyrimidine, pyrrole or pyrrolidine.
  • lipid as used herein means a substance that is soluble in organic solvents and includes, but is not limited to, oils, fats, sterols, triglycerides, fatty acids, phospholipids, and the like.
  • the lipid can be selected from the group consisting of fatty acids, fatty acid derivatives such as chlorides or esters, fatty alcohols, sterol lipids, glycerolipids (e.g., monoglycerides, diglycerides, and triglycerides), phospholipids, glycerophospholipids, sphingolipids, prenol lipids, saccharolipids, polyketides, and any combination thereof.
  • the lipid can be a polyunsaturated fatty acid or alcohol.
  • polyunsaturated fatty acid or “polyunsaturated fatty alcohol” as used herein means a fatty acid or alcohol with two or more carbon-carbon double bonds in its hydrocarbon chain.
  • the lipid can also be a highly unsaturated fatty acid or alcohol.
  • highly polyunsaturated fatty acid or “highly polyunsaturated fatty alcohol” as used herein means a fatty acid or alcohol having at least 18 carbon atoms and at least 3 double bonds.
  • the lipid can be an omega-3 fatty acid.
  • omega-3 fatty acid as used herein means a polyunsaturated fatty acid whose first double bond occurs at the third carbon-carbon bond from the end opposite the acid group.
  • the lipid can be selected from the group consisting of cholesterol; 1,3-Propanediol Dicaprylate/Dicaprate; 10-undecenoic acid; 1- dotriacontanol; 1-heptacosanol; 1-nonacosanol; 2-ethyl hexanol; Androstanes; Arachidic acid; Arachidonic acid; arachidyl alcohol; Behenic acid; behenyl alcohol; Capmul MCM CIO; Capric acid; capric alcohol; capryl alcohol; Caprylic acid; Caprylic/Capric Acid Ester of Saturated Fatty Alcohol C12-C18; Caprylic/Capric Triglyceride; Caprylic/Capric Triglyceride; Ceramide phosphorylcholine (Sphingomyelin, SPH); Ceramide phosphorylethanolamine (Sphingomyelin, Cer-PE); Ceramide phosphorylglycerol; Ceroplastic
  • glyceryl monocaprylate Capmul MCM C8 EP
  • Glyceryl Triacetate Glyceryl Tricaprylate
  • Glyceryl Tricaprylate/Caprate/Laurate Glyceryl Tricaprylate/Tricaprate
  • glyceryl 1,2- dipalmitate glyceryl 1,3-dipalmitate
  • glyceryl tripalmitate Tripalmitin
  • Henatriacontylic acid Heneicosyl alcohol; Heneicosylic acid; Heptacosylic acid; Heptadecanoic acid; Heptadecyl alcohol; Hexatriacontylic acid; isostearic acid; isostearyl alcohol; Lacceroic acid; Malawi acid; Lauryl alcohol; Lignoceric acid; lignoceryl alcohol; Linoelaidic acid; Linoleic acid; linolenyl alcohol; linoleyl alcohol; Margaric acid; Mead; Melissic acid;
  • neodecanoic acid neoheptanoic acid; neononanoic acid; Nervonic; Nonacosylic acid; Nonadecyl alcohol; Nonadecylic acid; Oleic acid; oleyl alcohol; Palmitic acid; Palmitoleic acid; palmitoleyl alcohol; Pelargonic acid; pelargonic alcohol; Pentacosylic acid; Pentadecyl alcohol; Pentadecylic acid; Phosphatidic acid (phosphatidate, PA); Phosphatidylcholine (lecithin, PC);
  • Phosphatidylethanolamine (cephalin, PE); Phosphatidylinositol (PI); Phosphatidylinositol bisphosphate (PIP2); Phosphatidylinositol phosphate (PIP); Phosphatidylinositol triphosphate (PIP3); Phosphatidylserine (PS); polyglyceryl-6-distearate; Pregnanes; Propylene Glycol Dicaprate; Propylene Glycol Dicaprylocaprate; Psyllic acid; recinoleaic acid; recinoleyl alcohol; Sapienic acid; soy lecithin; Stearic acid; Stearidonic; stearyl alcohol; Tricosylic acid; Tridecyl alcohol; Tridecylic acid; Triolein; Undecyl alcohol; undecylenic acid; Undecylic acid; Vaccenic acid; a-Lin
  • the lipid can be glyceryl 1,2-dipalmitate or glyceryl 1,3-dipalmitate. [0094] In some embodiments of any of the aspects, the lipid is a fatty acid derivative of
  • the fatty acid derivative is an acid chloride or an ester.
  • the adjuvant is a lipidated
  • the adjuvant is a lipidated oxoadinine.
  • the adjuvant is covalently linked to the lipid by a linker.
  • the adjuvant is covalently bound to an Ri group wherein Ri has the formula alkylene-L-R1-1, alkenylene-L-R1-1 or alkynylene-L-R1-1, wherein:
  • alkylene, alkenylene and alkynylene groups are optionally interrupted with one or more -O- groups, and preferably interrupted with one -O-group;
  • L is a bond or a functional linking group selected from the group consisting of -NHS(O) 2 -, - NHC(O)-, -NHC(S)-, -NHS(O) 2 NR 3 -, -NHC(O)NR 3 -, -NHC(S)NR 3 -, -NHC(O)0-, -0-, -S- and - S(O) 2 - ;
  • R 3 is selected from the group consisting of hydrogen and alkyl
  • R1-1 substituents are lipid moieties consisting of linear or branched aliphatic group having at least
  • R1-1 is -(CH 2 ) 1 oCH 3 , -(CH 2 ) 12 CH 3 , -(CH 2 ) 14 CH 3 , -(CH 2 ) 16 CH 3 ,
  • aliphatic group means a saturated or unsaturated linear or branched hydrocarbon group and includes alkyl, alkenyl and alkynyl groups.
  • alkyl refers to saturated or non-saturated non-aromatic hydrocarbon chains that may be a straight chain, branched chain and cyclic groups.
  • alkenyl refers to an alkyl that comprises at least one double bond.
  • alkynyl refers to an alkyl that comprises at least one triple bond. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms and alkynyl groups containing from 2 to 20 carbon atoms.
  • the lipid can be conjugated to adjuvant via a PEG linker.
  • PEG linkers can comprise from 1 to 15 repeats, e.g., repeats of a single PEG molecule. In some embodiments, the PEG linker can comprise from 3 to 9 repeats. In some embodiments, the PEG linker can comprise from 3 to 6 repeats. In some embodiments, the PEG linker can comprise 3 repeats. In some embodiments, the PEG linker can consist essentially of 3 repeats. In some embodiments, the PEG linker can consist of 3 repeats.
  • the length of the PEG linker can influence the activity of the adjuvant.
  • the PEG linker is 3 units or greater in length, e.g., 3 to 15, 3 to 9, 3 to 6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 units in length.
  • the PEG linker is 3 units or greater in length, e.g., 3 to 15, 3 to 9, 3 to 6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 units in length.
  • the PEG linker is 6 units or greater in length, e.g., 6 to 15, 6 to 9, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 units in length. In some embodiments of any of the aspects, wherein the adjuvant is desired to increase TNFa production, the PEG linker is 6 units or greater in length, e.g., 6 to 15, 6 to 9, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 units in length.
  • the PEG linker is 3, 4, or 5 units in length. In some embodiments of any of the aspects, wherein the adjuvant is desired to increase IFNa production but not TNFa, the PEG linker is 3, 4, or 5 units in length.
  • the adjuvant is 3M-052 (Formula VII below).
  • 3M-052 related compounds, and methods of making the same are described, e.g., in US Patent 7,799,800; which is incorporated by reference herein in its entirety.
  • the adjuvant is a lipidated
  • the adjuvant is lipidated R-848, lipidated 3M-002, lipidated 3M-013, lipidated 3M003, lipidated R837, lipidated gardiquimod, or lipidated CL097. In some embodiments of any of the aspects, the adjuvant is a lipidated thiazoquinoline.
  • the adjuvant can comprise both lipidated and unlipidated adjuvant molecules.
  • the adjuvant can be a
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is not conjugated to an antigen.
  • conjugated refers to the attachment of at least two entities to form one entity.
  • the joining of the two entities can be direct (e.g., via covalent or non-covalent bonds) or indirect (e.g., via linkers etc.). Conjugation can be by means of linkers, chemical modification, peptide linkers, chemical linkers, covalent or non- covalent bonds, or protein fusion or by any means known to one skilled in the art.
  • the joining can be permanent or reversible.
  • linker refers to any means, entity or moiety used to join two or more entities.
  • Linker moieties include, but are not limited to, chemical linker moieties, or for example a peptide linker moiety.
  • an "antigen” is a molecule that is specifically bound by a B cell receptor (BCR), T cell receptor (TCR), and/or antibody, thereby activating an immune response.
  • An antigen can be pathogen-derived, or originate from a pathogen.
  • An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof.
  • the term "antigenic determinant” refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.
  • the at least one antigen is comprised by a vaccine.
  • the vaccine is an attenuated vaccine.
  • Attenuated vaccines comprise weakened or compromised versions or variants of a disease- causing microbe.
  • Attenuated vaccines can include mutated or engineered strains of a microbe and/or strains which have been passaged in culture, thereby resulting in a loss of pathogenicity.
  • the vaccine can be a subunit vaccine, including a recombinant subunit vaccine.
  • a subunit vaccine does not comprise entire disease- causing microbes, but only a subset of antigens obtained from or derived from the disease- causing microbe.
  • a subunit vaccine can comprise multiple different antigens.
  • Subunit vaccines in which the antigens are produced via recombinant technologies are termed recombinant subunit vaccines.
  • the at least one antigen is comprised by a conjugate vaccine.
  • conjugate vaccines polysaccharides from a disease-causing microbe (e.g., polysaccahrides found on the surface of the microbe) are administered in combination with (e.g., conjugated to) an antigen which the patient's immune system already recognizes or which the patient's immune system will readily respond to. This increases the patient's response to the polysaccharides and provides increased protection against live versions of the disease-causing microbe.
  • the antigen is a polysaccharide.
  • Exemplary, non-limiting vaccines suitable for use in the methods and compositions described herein can include a pneumococcal vaccine; a hepatitis B (HBV) vaccine; an acellular pertussis (aP) vaccine; a diphtheria tetanus acellular pertussis (DTaP) vaccine; a hepatitis A (HAV) vaccine; a meningococcal (MV) vaccine; and/or pneumococcal conjugate vaccine (PCV)13.
  • HBV hepatitis B
  • aP acellular pertussis
  • DTaP diphtheria tetanus acellular pertussis
  • HAV hepatitis A
  • MV meningococcal
  • PCV pneumococcal conjugate vaccine
  • multiple antigens are administered.
  • multiple vaccines are administered.
  • the method described herein can further comprise administering a second adjuvant, e.g., sequentially or concurrently with the adjuvant comprising an agonist of TLR7 and/or TLR8.
  • the second adjuvant can be alum.
  • the antigen is bound to, adsorbed to, or conjugated to alum.
  • the vaccine can comprise alum. In some embodiments of any of the aspects, the vaccine is alum-adjuvanted.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 can be absorped onto alum. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 can be alum-absorped.
  • an adjuvant comprising an agonist of TLR7 and/or TLR8 surprisingly induces superior immune responses in newborns.
  • the adjuvant comprising an agonst of TLR7 and/or TLR8 can be administered to newborns and young patients, e.g., those of an age in which traditional adjuvants fail to produce a sufficient immune response.
  • the subject is a human subject.
  • the subject is a human infant at the time of administration.
  • the subject is a human of less than about 28 days of age at the time of administration.
  • the subject is a human of less than 28 of age days at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than about 4 days of age at the time of administration. In some embodiments of any of the aspects, the subject is a human of less than 4 days of age at the time of administration, e.g., less than 4 days, less than 3 days of age, less than 2 days of age, or less than 1 day of age. In some embodiments of any of the aspects, the administration occurs at the time of birth of the subject, e.g., during the perinatal period, during delivery, immediately following delivery, during transition, or during post-birth procedures. As used here, "perinatal period,” when used in reference to human subjects, refers to a period beginning at 22 completed weeks (154 days) of gestation and ends seven completed days after birth.
  • the methods described herein can further comprise at least a second administration of the adjuvant comprising an agonst of TLR7 and/or TLR8, or the adjuvant comprising an agonst of TLR7 and/or TLR8 and the antigen.
  • the adjuvant comprising an agonst of TLR7 and/or TLR8 (or the adjuvant and the antigen) is administered multiple times, the first administration occurs when the subject is less than about 28 days of age.
  • the first administration occurs when the subject is less than 28 days of age. In some embodiments of any of the aspects, wherein the adjuvant comprising an agonst of TLR7 and/or TLR8 (or the adjuvant and the antigen) is administered multiple times, the first administration occurs when the subject is less than about 1 day of age.
  • the first administration occurs when the subject is less than 1 day of age. In some embodiments of any of the aspects, wherein the adjuvant comprising an agonst of TLR7 and/or TLR8 (or the adjuvant and the antigen) is administered multiple times, the first administration occurs at the birth of the subject.
  • the first and/or second administration occurs when the subject is less than about 6 months of age. In some embodiments of any of the aspects, the first and/or second administration occurs when the subject is less than 6 months of age. In some embodiments of any of the aspects, the first and/or second administration occurs when the subject is less than about 28 days of age. In some embodiments of any of the aspects, the first and/or second administration occurs when the subject is less than 28 days of age. In some embodiments of any of the aspects, the first and/or second administration occurs when the subject is from about 28 days to about 6 months of age. In some embodiments of any of the aspects, the first and/or second administration occurs when the subject is from 28 days to 6 months of age.
  • the first and second administrations occur when the subject is less than about 6 months of age. In some embodiments of any of the aspects, the first and second administrations occur hen the subject is less than 6 months of age. In some embodiments of any of the aspects, the first and second administrations occur when the subject is less than about 28 days of age. In some embodiments of any of the aspects, the first and second administrations occur when the subject is less than 28 days of age. In some embodiments of any of the aspects, the first and second administrations occur when the subject is from about 28 days to about 6 months of age. In some embodiments of any of the aspects, the first and second administrations occur when the subject is from 28 days to 6 months of age.
  • the second administration occurs within about 28 days of the first administration. In some embodiments of any of the aspects, the second administration occurs within 28 days of the first administration.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered to a human infant. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered to a human newborn.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in the same formulation. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in the same formulation. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in the same formulation. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in different fomulations. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in different fomulations. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in different fomulations. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in different fomulations at the same time and in substantially the same location. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in different fomulations at different times. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 and the antigen (and optionally a second adjuvant) are administered in different fomulations and substantially at the same location.
  • compositions and methods described herein can be administered to a subject in need of vaccination, immunization, and/or stimulation of an immune response.
  • the methods described herein comprise administering an effective amount of compositions described herein, e.g. to a subject in order to stimulate an immune response or provide protection against the relevant pathogen the antigen was derived from.
  • Providing protection against the relevant pathogen is stimulating the immune system such that later exposure to the antigen (e.g., on or in a live pathogen) triggers a more effective immune response than if the subject was naive to the antigen. Protection can include faster clearance of the pathogen, reduced severity and/or time of symptoms, and/or lack of development of disease or symptoms.
  • compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or topical, administration. Administration can be local or systemic. In some embodiments of any of the aspects, the administration can be intramuscular or subcutaneous.
  • the term "effective amount” as used herein refers to the amount of adjuvant needed to stimulate the immune system, or in combination with an antigen, to provide a protective effect against subsequent infections, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the term "therapeutically effective amount” therefore refers to an amount of the adjuvant (and optionally, the antigen) that is sufficient to provide a particular immune stimulatory effect when administered to a typical subject.
  • An effective amount as used herein, in various contexts would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slowing the progression of a symptom of the disease), or prevent a symptom of the disease. Thus, it is not generally practicable to specify an exact "effective amount”. However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a composition which achieves a half-maximal inhibition of symptoms or induction of desired responses) as determined in cell culture, or in an appropriate animal model.
  • IC50 i.e., the concentration of a composition which achieves a half-maximal inhibition of symptoms or induction of desired responses
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for antibody titers, among others.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the technology described herein relates to a pharmaceutical composition comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 as described herein, and optionally a pharmaceutically acceptable carrier.
  • the active ingredients of the pharmaceutical composition comprises an adjuvant comprising an agonist of TLR7 and/or TLR8 as described herein.
  • the active ingredients of the pharmaceutical composition consist essentially of an adjuvant comprisiing agonist of TLR7 and/or TLR8 as described herein.
  • the active ingredients of the pharmaceutical composition consist of an adjuvant comprising an agonist of TLR7 and/or TLR8 as described herein.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media.
  • the use of such carriers and diluents is well known in the art.
  • Some non-limiting examples of materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent, e.g. an adjuvant comprising an agonist of TLR7 and/or TLR8 as described herein.
  • the pharmaceutical composition comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS -type dosage forms and dose- dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms of an adjuvant as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from underdosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • the adjuvant can be administered in a sustained release formulation.
  • Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos. : 3,845,770; 3,916,899;
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
  • active ingredients for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
  • OROS ® Alza Corporation, Mountain View, Calif. USA
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
  • an effective dose of a composition comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 as described herein can be administered to a patient once.
  • an effective dose of a composition comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 can be administered to a patient repeatedly.
  • subjects can be administered a therapeutic amount of a composition comprising an adjuvant comprising an agonist of TLR7 and/or TLR8, such as, e.g.
  • 0.1 mg/kg 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from about 0.001 mg per kilogram of a subject' body mass to about 10.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at dose of from 0.001 mg per kilogram of a subject's body mass to 10.0 mg per kilogram of the subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from about 0.01 mg per kilogram of a subject's body mass to about 10.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at dose of from 0.01 mg per kilogram of a subject's body mass to 10.0 mg per kilogram of the subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from about 0.01 mg per kilogram of a subject's body mass to about 5.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at dose of from 0.01 mg per kilogram of a subject's body mass to 5.0 mg per kilogram of the subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from about 0.05 mg per kilogram of a subject's body mass to about 5.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at dose of from 0.05 mg per kilogram of a subject's body mass to 5.0 mg per kilogram of the subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from about 0.01 mg per kilogram of a subject's body mass to about 1.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from 0.01 mg per kilogram of a subject's body mass to 1.0 mg per kilogram of the subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from about 0.05 mg per kilogram of a subject's body mass to about 0.5 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of from 0.05 mg per kilogram of a subject's body mass to 0.5 mg per kilogram of the subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of about 0.1 mg per kilogram of a subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered at a dose of 0.1 mg per kilogram of a subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is alum-absorbed and administered at a dose of from about 0.01 mg per kilogram of a subject's body mass to about 10.0 mg per kilogram of the subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is alum-absorbed and administered at a dose of from 0.01 mg per kilogram of a subject's body mass to 10.0 mg per kilogram of the subject's body mass.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 is alum-absorbed and administered at a dose of about 0.01 mg per kilogram of a subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is alum-absorbed and administered at a dose of 0.01 mg per kilogram of a subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is alum-absorbed and administered at a dose of about 10.0 mg per kilogram of a subject's body mass. In some embodiments of any of the aspects, the adjuvant comprising an agonist of TLR7 and/or TLR8 is alum-absorbed and administered at a dose of 10.0 mg per kilogram of a subject's body mass.
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the adjuvant and/or the antigen.
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses over a period of weeks or months.
  • the dosage ranges for the administration of an adjuvant comprising an agonist of TLR7 and/or TLR8 according to the methods described herein depend upon, for example, the form of the adjuvant, its potency, and the extent to which symptoms, markers, or indicators of a response described herein are desired to be induced, for example the percentage inducation desired for an immune response.
  • the dosage should not be so large as to cause adverse side effects, such as inflammatory responses.
  • the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • the efficacy of the adjuvant comprising an agonist of TLR7 and/or TLR8 in, e.g. to induce a response as described herein can be determined by the skilled clinician.
  • a treatment is considered "effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted signs or symptoms are improved, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate.
  • Immune responses can be detected by a variety of methods known to those skilled in the art, including but not limited to, antibody production, cytotoxicity assay, proliferation assay and cytokine release assays.
  • samples of blood can be drawn from the immunized mammal and analyzed for the presence of antibodies against the antigen administered in the respective vaccine and the titer of these antibodies can be determined by methods known in the art.
  • Efficacy of an agent can be determined by assessing physical indicators of a desired response, (e.g.
  • Efficacy can be assessed in animal models of a condition described herein, for example immunization of monkeys. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.
  • In vitro and animal model assays are provided herein which allow the assessment of a given dose of an adjuvant and/or antigen.
  • the effects of a dose of adjuvant can be assessed by measuring the antibody titers or cytokine production.
  • the efficacy of a given dosage combination can also be assessed in an animal model, e.g. immunization of infant or newborn monkeys as described in the Examples herein.
  • kits comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 and optionally at least one antigen.
  • the adjuvant and antigen are not conjugated to each other.
  • the adjuvant and antigen can be present in the same formulation of the kit or in separate formulations of the kit, e.g., for separate administration or for mixing prior to administration.
  • kits are any manufacture (e.g., a package or container) comprising at least one reagent, e.g., an adjuvant comprising an agonist of TLR7 and/or TLR8, the manufacture being promoted, distributed, or sold as a unit for performing the methods described herein.
  • the kits described herein can optionally comprise additional components useful for performing the methods described herein.
  • the kit can comprise fluids and compositions (e.g., buffers, needles, syringes etc.) suitable for performing one or more of the administrations according to the methods described herein, an instructional material which describes
  • the kit may comprise an instruction leaflet.
  • an "immune response” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus (e.g., to an adjuvant).
  • a cell of the immune system such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil.
  • a stimulus e.g., to an adjuvant.
  • the response is specific for a particular antigen (an "antigen-specific response”), and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor.
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • T cell response such as a CD4+ response or a CD8+ response.
  • responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.
  • Stimulation of an immune response refers to an induction or increase of the immune response.
  • an immune response can be cytokine production by Thl cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of Thl antigen-specific neonatal CD4+ cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of Thl neonatal CD4+ cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of Thl neonatal cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of neonatal CD4+ cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of Thl CRM-197-specific neonatal CD4+ cells.
  • the immune response is an increase in the IgG2a/c subclass. In some embodiments of any of the aspects, the immune response is an increase in the IgG2a/c subclass and the adjuvant comprising an agonist of TLR7 and/or TLR8 is absorbed to alum.
  • An immune response to an antigen can be the development in a subject of a humoral and/or a cell-mediated immune response to molecules present in the antigen or vaccine composition of interest.
  • a “humoral immune response” is an antibody-mediated immune response and involves the induction and generation of antibodies that recognize and bind with some affinity for the antigen in the immunogenic composition of the invention, while a “cell-mediated immune response” is one mediated by T-cells and/or other white blood cells.
  • a “cell-mediated immune response” is elicited by the presentation of antigenic epitopes in association with Class I or Class II molecules of the major histocompatibility complex (MHC), CD1 or other non-classical MHC-like molecules. This activates antigen- specific CD4+ T helper cells or CD8+ cytotoxic lymphocyte cells ("CTLs").
  • MHC major histocompatibility complex
  • CTLs have specificity for peptide antigens that are presented in association with proteins encoded by classical or non-classical MHCs and expressed on the surfaces of cells. CTLs help induce and promote the intracellular destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide or other antigens in association with classical or non-classical MHC molecules on their surface.
  • a “cell-mediated immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells. The ability of a particular antigen or composition to stimulate a cell-mediated immune response
  • immunological response may be determined by a number of assays, such as by
  • lymphoproliferation (lymphocyte activation) assays CTL cytotoxic cell assays, by assaying for T-lymphocytes specific for the antigen in a sensitized subject, or by measurement of cytokine production by T cells in response to re-stimulation with antigen.
  • assays are well known in the art. See, e.g., Erickson et al. (1993) J. Immunol. 151 :4189-4199; and Doe et al. (1994) Eur. J. Immunol. 24:2369-2376.
  • treatment means any one or more of the following: (i) the prevention of infection or re-infection, as in a traditional vaccine, (ii) the reduction in the severity of, or, in the elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen or disorder in question.
  • treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
  • prophylactic treatment is the preferred mode.
  • compositions and methods that treat, including prophylactically and/or therapeutically immunize, a host animal against a microbial infection (e.g., a bacterium or virus).
  • a microbial infection e.g., a bacterium or virus.
  • the methods of the present invention are useful for conferring prophylactic and/or therapeutic immunity to a subject.
  • the methods of the present invention can also be practiced on subjects for biomedical research applications.
  • immunologically effective amount of the adjuvant comprising an agonist of TLR7 and/or TLR8 is administered.
  • immunologically effective amount refers to the amount of the antigen or immunogenic composition sufficient to elicit an immune response, either a cellular (T-cell) or humoral (B-cell or antibody) response, or both, as measured by standard assays known to one skilled in the art.
  • the term "vaccine composition” used herein is defined as composition used to elicit an immune response against an antigen within the composition in order to protect or treat an organism against disease.
  • the vaccine composition is a suspension of attenuated or killed microorganisms (e.g., viruses, bacteria, or rickettsiae), or of antigenic proteins derived from them, administered for prevention, amelioration, or treatment of infectious diseases.
  • the terms "vaccine composition” and “vaccine” are used interchangeably.
  • the term "newborn” refers to an infant from the time of birth through the 28th day of life. In some embodiments of any of the aspects, the newborn is a human infant. In the embodiment that the newborn is a premature birth, the 28 th day is extended to include the number of days of premature birth.
  • infant refers to a young from the time of birth to one year of age.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%), or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100%) as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a "increase” is a statistically significant
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subj ect is a mammal, e.g., a primate, e.g., a human.
  • the terms, "individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of immunization and immune response.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. susceptibility to infection) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a condition in need of treatment e.g. susceptibility to infection
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • Protein and “polypeptide” are often used in reference to relatively large
  • polypeptides whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • protein and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid,
  • the nucleic acid can be either single-stranded or double-stranded.
  • a single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single- stranded nucleic acid not derived from any double-stranded DNA.
  • the nucleic acid can be DNA.
  • the nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA.
  • a polypeptide, nucleic acid, or cell as described herein can be engineered.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be “engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.
  • progeny of an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • the term "pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term "corresponding to” refers to an atom or group at the specified or enumerated position in a molecule, or an atom or group that is equivalent to a specified or enumerated atom or group in a second molecule. Equivalent specified or
  • enumerated atoms/groups can be determined by one of skill in the art, e.g., by idenitifying shared core structures or formulas.
  • a method of immunizing a subject comprising administering to the subject i) an adjuvant comprising an agonist of TLR7 and/or TLR8; and ii) at least one antigen;
  • the adjuvant and the at least one antigen are not conjugated to each other.
  • the adjuvant is selected from the group consisting of:
  • RNA single sstranded (ss) RNA
  • imidazoquinoline a thiazoquinoline and a benzazepine.
  • a pneumococcal vaccine a hepatitis B (HBV) vaccine; an acellular pertussis (aP) vaccine; a diphtheria tetanus acellular pertussis (DTaP) vaccine; a hepatitis A (HAV) vaccine; and a meningococcal (MV) vaccine.
  • HBV hepatitis B
  • aP acellular pertussis
  • DTaP diphtheria tetanus acellular pertussis
  • HAV hepatitis A
  • MV meningococcal
  • the vaccine is pneumococcal conjugate vaccine (PCV)13.
  • a method of stimulating an immune response of a subject comprising administering to the human an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • ss single sstranded RNA
  • imidazoquinoline a thiazoquinoline
  • benzazepine a single sstranded RNA
  • compositions for use in immunizing a subject or stimulating an immune response in a subject comprising an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • compositions further comprises at least one antigen, wherein the adjuvant and the at least one antigen are not conjugated to each other.
  • a composition or kit comprising a first formulation comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 and a second formulation comprising at least one antigen, wherein the formulations are for use in immunizing a subject or stimulating an immune response in a subject.
  • a kit comprising an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • kit of paragraph64 further comprising at least one antigen.
  • RNA single sstranded (ss) RNA
  • imidazoquinoline a thiazoquinoline and a benzazepine.
  • composition or kit of paragraph73 wherein the antigen is a polysaccharide.
  • a vaccine selected from the group consisting of: a pneumococcal vaccine; a hepatitis B (HBV) vaccine; an acellular pertussis (aP) vaccine; a diphtheria tetanus acellular pertussis (DTaP) vaccine; a hepatitis A (HAV) vaccine; and a meningococcal (MV) vaccine.
  • composition or kit of paragraph76, wherein the vaccine is pneumococcal conjugate vaccine (PC V)l 3.
  • composition or kit of any of paragraphs 61-78,further comprising a second adjuvant comprising a second adjuvant.
  • composition or kit of any of paragraphs 61-89, wherein the subject is further
  • composition or kit of paragraph90 wherein the first administration occurs when the subject is less than 1 day of age.
  • administration occur when the subject is less than 6 months of age.
  • administration occur when the subject is less than 28 days of age.
  • administration occur when the subject is from 28 days to 6 months of age.
  • a method of immunizing a subject comprising administering to the subject i) an adjuvant comprising an agonist of TLR7 and/or TLR8; and ii) at least one antigen;
  • RNA a single sstranded (ss) RNA; an imidazoquinoline; a thiazoquinoline; an oxoadinine; and a benzazepine.
  • n is from 0 to 20
  • R is R is selcted from H, Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl- 6alkyl, C3-6cycloalkylCl-6alkylamino, C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl- 6alkyl, Cl-6alkoxyCl-6alkylamino and Cl-6alkoxyCl-6alkoxy; wherein the Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl-6alkyl, C3-6cycloalkylCl-6alkylamino, 20 C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl-6alkyl, Cl-6alkoxyCl-6alkylamino or Cl- 6alkoxyCl-6alkoxy is branched or unbranched and optionally terminally substituted with a hydroxyl, amino, thio, hydrazino
  • X is a phospholipid, lipid, lipidation, and/or PEG moiety.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a com ound having the structure of Formula X:
  • the adjuvant comprising an agonist TLR7 and/or TLR8 comprises a compound having the structure of Formula XI:
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound selected from the group consisting of: 3M- 052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; and CRX-748.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 further comprises a lipid moiety
  • the adjuvant further comprises a phosphorylation or phospholipid moiety.
  • a pneumococcal vaccine a hepatitis B (HBV) vaccine; an acellular pertussis (aP) vaccine; a diphtheria tetanus acellular pertussis (DTaP) vaccine; a hepatitis A (HAV) vaccine; and a meningococcal (MV) vaccine.
  • HBV hepatitis B
  • aP acellular pertussis
  • DTaP diphtheria tetanus acellular pertussis
  • HAV hepatitis A
  • MV meningococcal
  • a method of stimulating an immune response of a subject comprising administering to the human an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • the method of paragraph 50 wherein the immune response is T helper 1- cytokine production.
  • RNA a single sstranded (ss) RNA; an imidazoquinoline; a thiazoquinoline; an oxoadinine; and a benzazepine.
  • n is from 0 to 20
  • R is R is selcted from H, Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl- 6alkyl, C3-6cycloalkylCl-6alkylamino, C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl- 6alkyl, Cl-6alkoxyCl-6alkylamino and Cl-6alkoxyCl-6alkoxy; wherein the Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl-6alkyl, C3-6cycloalkylCl-6alkylamino, 20 C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl-6alkyl, Cl-6alkoxyCl-6alkylamino or Cl- 6alkoxyCl-6alkoxy is branched or unbranched and optionally terminally substituted with a hydroxyl, amino, thio, hydrazino
  • X is a phospholipid, lipid, lipidation, and/or PEG moiety.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound selected from the group consisting of: 3M- 052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; and CRX-748.
  • the adjuvant comprising an agonist of TLR7 and/or TLR8 further comprises a lipid moiety.
  • the adjuvant further comprises a phosphorylation or phospholipid moiety.
  • compositions for use in immunizing a subject or stimulating an immune response in a subject comprising an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • composition of paragraph 90 wherein the composition further comprises at least one antigen, wherein the adjuvant and the at least one antigen are not conjugated to each other.
  • a composition or kit comprising a first formulation comprising an adjuvant comprising an agonist of TLR7 and/or TLR8 and a second formulation comprising at least one antigen, wherein the formulations are for use in immunizing a subject or stimulating an immune response in a subject.
  • kits comprising an adjuvant comprising an agonist of TLR7 and/or TLR8.
  • kit of paragraph 93 further comprising at least one antigen.
  • n is from 0 to 20 ,
  • R is R is selcted from H, Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl- 6alkyl, C3-6cycloalkylCl-6alkylamino, C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl- 6alkyl, Cl-6alkoxyCl-6alkylamino and Cl-6alkoxyCl-6alkoxy; wherein the Cl-6alkyl, Cl-6alkylamino, Cl-6alkoxy, C3-6cycloalkylCl-6alkyl, C3-6cycloalkylCl-6alkylamino, 20 C3-6cycloalkylCl-6alkoxy, Cl-6alkoxyCl-6alkyl, Cl-6alkoxyCl-6alkylamino or Cl- 6alkoxyCl-6alkoxy is branched or unbranched and optionally terminally substituted with a hydroxyl, amino, thio, hydrazino
  • X is a phospholipid, lipid, lipidation, and/or PEG moiety.
  • composition or kit of any of paragraphs 90-97, wherein the adjuvant comprising an agonist of TLR7 and/or TLR8 comprises a compound selected from the group consisting of: 3M-052; CRX-648; CRX-649; CRX-664; CRX-672; CRX-677; and CRX-748.
  • TLR7 and/or TLR8 comprises CRX-649.
  • TLR7 and/or TLR8 further comprises a lipid moiety
  • composition or kit of paragraph 105 wherein the PEG linker comprises from 3 to 9 repeats of PEG.
  • composition or kit of paragraph 105 wherein the PEG linker comprises 3 repeats of PEG.
  • composition or kit of any of paragraphs 90-109, wherein the antigen is
  • subunit vaccine comprised by a subunit vaccine or recombinant subunit vaccine.
  • composition or kit of any of paragraphs 90-109, wherein the antigen is
  • composition or kit of paragraph 112 wherein the antigen is a polysaccharide.
  • composition or kit of any of paragraphs 90-114, wherein the antigen is
  • a vaccine selected from the group consisting of:
  • a pneumococcal vaccine a hepatitis B (HBV) vaccine; an acellular pertussis (aP) vaccine; a diphtheria tetanus acellular pertussis (DTaP) vaccine; a hepatitis A (HAV) vaccine; and a meningococcal (MV) vaccine.
  • HBV hepatitis B
  • aP acellular pertussis
  • DTaP diphtheria tetanus acellular pertussis
  • HAV hepatitis A
  • MV meningococcal
  • composition or kit of paragraph 115, wherein the vaccine is pneumococcal conjugate vaccine (PCV)13.
  • PCV pneumococcal conjugate vaccine
  • composition or kit of any of paragraphs 90-116, wherein the vaccine is alum- adjuvanted is alum- adjuvanted.
  • composition or kit of paragraph 119 wherein the adjuvant comprising the agonist of TLR7 and/or TLR8 is absorped onto the alum.
  • composition or kit of any of paragraphs 90-131, wherein the adjuvant is formulation for administration intramuscularly or subcutaneously.
  • composition or kit of any of paragraphs 90-132 further comprising at least a second administration of the adjuvant and antigen.
  • the composition or kit of paragraph 133 formulated for the first administration to occur when the subject is less than 1 day of age.
  • composition or kit of paragraph 133 formulated for the first administration to occur at the birth of the subject.
  • composition or kit of paragraph 133 formulated for the first administration to occur when the subject is less than 28 days of age.
  • EXAMPLE 1 TLR7/8 Adjuvant Overcomes Newborn Hyporesponsiveness to Pneumococcal Conjugate Vaccine at birth
  • Infection is the most common cause of mortality in early life and immunization is the most promising biomedical intervention to reduce this burden.
  • newborns fail to optimally respond to most vaccines.
  • Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most Toll-like receptor (TLR) agonists, are functionally distinct.
  • TLR Toll-like receptor
  • 3M-052 is a locally-acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, that when properly formulated, can induce robust T helper 1 -cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the Alum-adjuvanted pneumococcal conjugate vaccine (PCV)13.
  • PCV pneumococcal conjugate vaccine
  • 3M-052 When admixed with PC VI 3 and administered intramuscularly on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Thl CRM-197-specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide (PnPS)-specific B cells as well as serotype-specific antibody titers and opsonophagocytic killing.
  • PnPS Streptococcus pneumoniae polysaccharide
  • opsonophagocytic killing Remarkably, a single birth dose of (PCV13 + 0.1 mg/kg 3M-052) induced PnPS-specific IgG responses that were -10 - 100 times greater than a single birth dose of PC VI 3 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life.
  • accelerated neonatal immunization strategies may be highly advantageous (3, 4) because: a) newborn vaccines achieve relatively high population penetration as birth is the most reliable point of health care contact worldwide (5), b) there is high risk of severe infection after very early life colonization, and c) reduced vaccine responses can occur after bacterial colonization (6, 7).
  • Adjuvantation is a key tool to enhance vaccine-induced immunity. Adjuvants can enhance, prolong, and modulate immune responses to vaccinal antigens to maximize protective immunity (8), and may potentially enable effective immunization in the very young (1).
  • responses of human newborn leukocytes to most adjuvants, including most Tolllike receptor (TLR) agonists (TLRAs) are functionally distinct (2).
  • Considerations in selecting a clinically relevant adjuvanted vaccine formulation include (a) minimizing systemic inflammation (9), that can occur with TLRAs included in soluble or, to a lesser extent, with TLRA adjuvant-conjugated nanoparticle-based formulations (10), and (b) ensuring activity towards the target population - not a forgone conclusion in newborns, given age-specific soluble and cellular factors (1) that shape distinct T helper (Th)- mediated immunity (11), potentially limiting immune responses to vaccines and pathogens (12, 13).
  • Th shape distinct T helper- mediated immunity
  • TLR7 and 8 those that most effectively activate human newborn leukocytes are agonists of TLR7 and 8, a sub-family of endosomal leukocyte pattern recognition receptors (PRRs) that recognize uridine-rich single stranded ribonucleic acid (RNA) molecules, as are found in viral RNA, and synthetic imidazoquinolines (IMQs) (14-16).
  • PRRs endosomal leukocyte pattern recognition receptors
  • IMQs imidazoquinolines
  • immunization schedule- is feasible and effective at birth (i.e., the first 24 hours of life), a key point of global healthcare contact during which the immune system is most distinct.
  • birth i.e., the first 24 hours of life
  • a rational vaccine design approach was undertaken, employing a TLR7/8A adjuvant.
  • 3M-052 a locally-acting lipidated IMQ TLR7/8A which can induce tumor- specific immunity by forming agonist depots for a gradual sustained release (17) was utilized.
  • Pneumococcus is an important pediatric pathogen comprised of > 92 different capsular polysaccharide serotypes that causes serious invasive disease, including meningitis, sepsis, otitis media, and pneumonia, and is responsible for -10% of worldwide deaths in children less than 5 years of age (19).
  • the poor efficacy of plain polysaccharide vaccines in young children prompted the development of PCVs that induce T cell-dependent mechanisms (20), with a recommended 3 to 4-dose schedule starting at 2 months of age (21).
  • PCV-induced protection may not be fully achieved until 6 - 12 months of life (18), and the inclusion of Alum, though safe and effective, appears to be Th2- polarizing (22) and results in a formulation that requires multiple doses prior to achieving protective Ab titers.
  • PCV pneumococcal conjugate vaccine
  • PCV7 immunization with a 3 -dose schedule starting at birth, induced protective serum Ab concentrations in human infants as early as 18 weeks (4.5 months), neonatal hypo-responsiveness was noted for several vaccine serotypes as compared to infants starting a 3-dose schedule of PCV7 at 2 months of life (22, 23).
  • 3M-052 induced robust Thl -cytokine production by human newborn and adult leukocytes in vitro, both alone and in synergy with Alum-adjuvanted PCV13.
  • 3M-052 Using a clinically relevant neonatal rhesus macaque model, it is demonstrated that, when admixed with PCV13 and administered intramuscularly, 3M-052 dramatically accelerated and enhanced neonatal B and T cell immune responses rapidly amplifying functional serotype- specific Ab titers to concentrations that correlate with protection after the first dose of a 3-dose series (Day of Life (DOL) 0, 28, and 56), without serious adverse effects.
  • DOL Day of Life
  • 3M-052 is a locally acting TLR7/8 Agonist
  • 3M-052 a TLR7/8A that bears a CI 8 lipid moiety (24) that serves to localize its action, was selected.
  • Rodent pharmacokinetic (PK) and pharmacodynamic (PD) studies were conducted to compare the IMQ TLR7/8A R848 (Resiquimod) and its lipidated congener 3M-052 (Fig. 1 A), that can be formulated in an oil-in-water (O/W) emulsion vehicle (Table 3).
  • PK differences were observable by measurement of R848 or 3M-052 serum drug levels determined by liquid chromatography-mass spectrometry (LC-MS/MS) pre- and post- a single intramuscular (IM, to quadriceps) or subcutaneous (SC, to scruff of neck) administration (Fig. IB).
  • LC-MS/MS liquid chromatography-mass spectrometry
  • IM intramuscular
  • SC subcutaneous
  • Fig. 3 A four cohorts of five rhesus macaques per cohort (Fig. 3 A) were immunized IM with saline (control), 3M-052 adjuvant alone (0.1 mg/kg 3M-052, or 40 ⁇ g per animal), a half dose of PCV13 alone, or PCV13 admixed with 3M-052 (PC VI 3 + 0.1 mg/kg 3M-052). All treatments began with a birth dose (DOL0), followed by booster doses at one (DOL28) and two months (DOL56) of life (Fig. 3B). Peripheral blood was collected at the indicated time-points to obtain plasma for an assay of anti-pneumococcal serotype Ab titers by polysaccharide-IgG binding microarray (Table 4).
  • TLR7/8A adjuvantation dramatically accelerates and enhances serotype-specific pneumococcal opsonophagocytic killing
  • PCV13 + 0.1 mg/kg 3M-052)-immunized rhesus macaques demonstrated functional Ab responses to all 13 PS serotypes contained in PCV13. Consistent with the striking observations seen for PnPS-specific IgG titers, functional Ab-mediated responses were dramatically accelerated in animals receiving a single dose of (PCV13 + 3M-052) - i.e., PCV adjuvanted with either dose of adjuvant. All animals receiving (PCV13 + 0.1 mg/kg 3M-052) demonstrated a robust functional Ab activity to 11 of the 13 serotypes tested by DOL28, with opsonization indices (OIs) -10 - 100 greater than PCV13 alone (Table 1).
  • 3M-052 has limited systemic activity in neonatal and infant primates
  • CBCs Complete blood counts
  • peripheral blood mononuclear cells were sorted to obtain highly pure populations of B cells, T cells and monocytes that were used for the evaluation of antigen specific vaccine-induced B- and T-cells in infant rhesus macaques by means of enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining assays, respectively.
  • ELISPOT enzyme-linked immunospot
  • 3M-052 enhances and accelerates activation of early life PnPS-specific B cells
  • ELISPOT indicates that 3M-052 accelerated and enhanced B cell activation (Fig. 23D). Indeed, addition of 3M-052 to PCV13 was associated with increased infiltration of CD68+ cells (i.e., monocytes/macrophages) into the injection site muscle (Fig. 24).
  • CD68+ cells i.e., monocytes/macrophages
  • BCG Bacille Calmette Guerin
  • 3M-052 as a lipidated TLR7/8A adjuvant that both alone and in synergy with Alum induced Thl -cytokine responses at birth, and that when administered with Alum-adjuvanted PCV13 in vivo dramatically accelerated and enhanced neonatal antigen- specific immunogenicity after a single immunization.
  • 3M-052 synergistically enhanced type II interferon and Thl -polarizing human cord blood cytokine production to PCV13 in vitro, and dramatically accelerated S. pneumoniae antigen-specific neonatal rhesus macaque B- and Thl-cell responses ex vivo.
  • 3M-052 to enhance and accelerate activation of anti-PnPs-IgG, PnPS-specific B cells, Thl-polarized CRM197-specific CD4+ T cells and synergistically activated type II IFN responses in vitro shares similarity with the immune polarizing effects of systemic viral infection (35) and signatures of bacterial viability (36), highlighting the potential of TLR7/8-triggered pathways to fundamentally shape immune responses (37), especially vaccinal antigen-specific-IFN ⁇ -producing T cells in early life (10).
  • adjuvanted vaccine development A key concern regarding adjuvanted vaccine development is reactogenicity, the propensity of a formulation to cause acute inflammatory events either locally - e.g., erythema, tenderness - or systemically as fever.
  • vaccine adjuvants are not licensed separately; rather, the adjuvant is a constituent of the licensed vaccine formulation. Therefore, as demonstrated in the assays described herein, adjuvants must be evaluated both alone and as a component of a vaccine formulation. To the extent that they reflect activity in vivo, development of reliable platforms for in vitro modeling may help exclude adjuvants with high potential to induce unacceptable reactogenicity in the very young (1, 4).
  • the longitudinal rhesus monkey experimental protocol (number P0184) was approved by the IACUC at Tulane University and performed at Tulane National Primate Research Center (TNPRC; Covington, LA). Additionally, peripheral blood samples from rhesus macaques were derived from New England Primate Research Center (NEPRC) (Southborough, MA) and used under Harvard University IACUC approval (protocol number 04936).
  • 3M-052 and R848 serum drug levels were determined by LC-MS/MS pre- or post-dose with a lower limit of quantification (LLQ) of 0.84 and 3.2 pmol/ml, respectively.
  • serum TNF concentrations were measured by ELISA at the indicated times pre- or post-dose, with a LLQ of 31 pg/ml.
  • mRNA expression in draining lymph nodes and spleen post- administration were determined by quantitative RT-PCR (Applied Biosystems; Carlsbad, CA) as described previously(24) and represented as relative fold-change expression (i.e., treatment relative expression/naive relative expression).
  • HA hemagglutinin
  • 6 - 8 week old male Balb/c mice were immunized by subcutaneous injection (scruff of neck) with recombinant influenza A hemagglutinin (HA, 10 ⁇ g) alone or in combination with 0.01, 0.03, 0.1, 0.3, or 1 mg/kg 3M- 052, or in combination with Alum, three times (prime, boost, boost) 14 days apart.
  • HA-specific serum Ig levels measured by ELISA on day 77, 21 days post-final immunization as described previously (24).
  • TLR agonists and multi-analyte assays were used at the concentrations noted in the figure legends.
  • R848 (TLR7/8) was purchased from InvivoGen (San Diego, CA). All TLR7/8As and emulsions used in both in vitro and in vivo studies were verified to be free of endotoxin ( ⁇ 1 EU/ml) by the Limulus amoebocyte lysate (LAL) assay per the manufacturer's instructions (Charles River; Wilmington, MA).
  • LAL Limulus amoebocyte lysate
  • Cytokine and chemokine expression profiles in cell culture supernatants and peripheral blood plasma were measured using customized Milliplex human and non-human primate cytokine/chemokine magnetic bead panels (Millipore), respectively. Assays were analyzed on the Luminex® 100/200TM System employing xPOTENT® software (Luminex; Austin, TX) and Millipore Milliplex Analyst (version 3.5.5.0).
  • Vaccine formulation The point-of-use mixed vaccine formulation consisted of 2 components. Firstly, an oil-in-water emulsion (OAV) consisting of a pH 6 citrate buffer, soybean oil, and surfactants that contains 0.04 - 0.4 mg/ml of N-[4- [(4-amino-2-butyl-lH-imidazo[4,5- c]quinolin-l-yl)oxy]butyl]octadecanamide (3M-052) (24) (3M Drug Delivery Systems Division, 3M Center; St. Paul, MN). Concentrations of 3M-052 OAV emulsion preparations were confirmed by high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the 3M-052 OAV emulsion formulations were sterile filtered, aliquoted into sterile 2 ml serum vials sealed with rubber septa, and stored at 2 - 8°C until use. Dual agonist activity of 3M-052 was confirmed using FEK293 cells stably expressing either human TLR7 or TLR8 (24). The dosing range of 3M-052 was approximately 4 - 40 ⁇ g (0.01 - 0.1 mg/kg; 400 g birth weight) (Table 3). Secondly, one-half of the recommended human infant dose of the Pneumococcal 13-valent Conjugate Vaccine
  • Murine TLR8 is divergent from human and monkey TLR8, and mice mount distinct immune responses to TLR7/8As and TLR8As (46).
  • Rhesus macaques are likely a relevant animal model for predicting TLR8 adjuvant responses in human infants (26, 47).
  • TLR/TIR orthologues have been identified within the Rhesus macaque (M. mulata) genome, with an overall mean amino acid identity of 96.7% to their corresponding human TLR/TIR sequences, compared with 87.4% to mouse TLR/TIR sequences (25).
  • TLR8 The most highly conserved TLR/TIR is TLR8, which demonstrates 98.6% amino acid identity to human TLR8.
  • TLR8 in Rhesus macaques and humans is highly conserved in terms of its predicted distribution pattern of extracellular LRRs.
  • Rhesus macaques are also well suited for our study because: (a) adult rhesus macaques have demonstrated humanlike responses to TLR7/8As in vivo (48-50), (b) both infant and adult rhesus macaques demonstrate human-like TLR7/8A-induced cytokine responses in vitro (14), and (c) like humans, infant rhesus macaques respond immunologically to conjugated, but not to unconjugated polysaccharides in vivo.
  • Animals were group-housed in dam/infant pairs with a maximum of 4 pairs (8 animals) together. All animals received standard environmental enrichment, including
  • Photograph 1 the tattoo number (to avoid false attribution of pictures to animals); Photograph 2: both ventral thighs; Photographs 3 and 4: each ventral thigh individually; and Photographs 5 - 8: each thigh individually from medial and lateral aspects. If local erythema (redness) or swelling were noted, a higher magnification photograph was taken of the area. Finally, the newborn underwent IM vaccination as outlined above. After DOL70, physical exam alone (i.e., without photography) was conducted according to the same schedule, up to 1 year of life.
  • IM ketamine hydrochloride 10 mg/kg
  • dexmedetomidine 7.5 - 15 ⁇ g/kg
  • IM tiletimine/zolazepam 8 mg/kg
  • atipamezole was administered IM as a reversal agent when dexmedetomidine was used.
  • the cranial aspect of the rear limb distal to the coxo- fem oral joint and proximal to the stifle were surgically prepped, a sterile fenestrated drape placed on the cranial aspect of the rear limb, and #15 scalpel blade used to make a 3 mm incision through the skin. Skin adjacent to the incision was undermined, and muscle tissue was exteriorized using sterile rat tooth forceps. Curved scissors were used to excise a 2mm length of superficial musculature.
  • the 2 mm cube muscle biopsies were obtained from the injection site (quadriceps muscle) prior to and 48 hours after each immunization (one in each thigh), and obtained in an alternating pattern (e.g. DOL0 left leg, DOL2 right leg, DOL30 left leg, DOL58 right leg).
  • Lymph node biopsies were obtained on DOL7 and 63, and followed a similar pattern of alternation.
  • Peripheral blood samples were drawn from each group at multiple time-points per Supplemental Table 2, including at DOL0 (pre-immunization), DOL7, 28, 30, 35, 56, 63, 90, 150, 180, 240, and 360. Serum and plasma samples were stored at -80 °C for subsequent immunogenicity assays.
  • Peripheral blood mononuclear cells (PBMCs) were isolated and stored in liquid Nitrogen.
  • Pneumococcal polysaccharides were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) or Statens Serum Institut (SSI; Copenhagen, Denmark) for Danish serotype designations 3, 4, 6B, 9V, 14, 18C, 19F, and 23F. These were individually spotted in each well (100 ⁇ g/ml coating
  • Serum was heat inactivated at 56 °C for 30 min prior to incubation with target pneumococcal bacterial strains (BEI Resources; VA, USA) for an additional 30 min.
  • the opsonophagocytic incubation reaction occurred at 56 °C for 30 min with baby rabbit serum (Pel-freez Biologicals, Rogers; AR, USA) as the complement source and human pro-myelocytic cell line HL-60 cells (ATCC) as the phagocytic cells.
  • opsonophagocytic incubation reaction mixtures were transferred to agar media to allow bacterial growth, digital images obtained, and surviving colonies enumerated using automated software (US National Institute of Standards and Technology (NIST) Integrated Colony Enumerator).
  • Opsonization titers were defined as the serum dilution that kills 50% of bacteria. The lowest detectable titer in the MOP A was 24, and therefore, samples identified as negative in the assay (i.e., samples having no functional activity detected) were assigned a titer of 12 (i.e., half the lowest limit of detection).
  • Anti-pneumococcal B cells in peripheral blood were enumerated in sorted B cell populations.
  • Sorted B cell populations were cultured at a concentration of 5 x 106/ml for 5 days at 37 °C, 5% C02 in RPMI media supplemented with Penicillin/Streptomycin, 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 1 ⁇ g/ml R848 (Invivogen; San Diego, CA, USA), 10 IU/ml IL-2 (R&D Systems; Minneapolis, MN, USA), and 8000 U/ml IFNy (Abeam; Cambridge, MA, USA).
  • FBS Invitrogen, Carlsbad, CA, USA
  • 1 ⁇ g/ml R848 Invivogen; San Diego, CA, USA
  • 10 IU/ml IL-2 R&D Systems; Minneapolis, MN, USA
  • 8000 U/ml IFNy Abeam; Cambridge, MA, USA.
  • ELISpot plates (Millipore) were coated either with a combination of 10 ⁇ g/ml anti-rhesus IgG-Fc and anti-rhesus IgM-Fc (Nordic Immunological Laboratories; Eindhoven, The Netherlands) in phosphate-buffered saline (PBS), or with a 10 ⁇ g/ml pool of the following pneumococcal polysaccharides: Danish designations 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A, 23F (ATCC), 7F, and 19F (SSI) in PBS. Plates were coated overnight at 4°C and blocked with RPMI 1640/10% FBS for 1 hour prior to plating of cells.
  • B cells were incubated for 16 hours on the coated and blocked ELISpotTM plate in RPMI supplemented with Penicillin/Streptomycin and 10%) FBS. Unless indicated otherwise, 10%> of each culture (-50,000 cells) was plated in anti-rhesus IgG/IgM-coated wells and 90%> of each culture (-450,000 cells) was plated in wells coated with polysaccharides. Secreted immunoglobulins were detected using horseradish peroxidase-conjugated goat-anti-rhesus immunoglobulin (Nordic Immunological Laboratories; Eindhoven, The Netherlands).
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • ELISpotTM analyzer Cellular Technology Limited, Shaker Heights; OH, USA.
  • the fraction of pneumococcal polysaccharide-specific B cells was quantified as the ratio of the spots detected in polysaccharide-coated wells to spots detected in immunoglobulin-coated wells, after correction for dilution.
  • CD4+ T cells Characterization of CRM197-specific CD4+ T cells.
  • Monocytes and T cells were sorted as described above. Sorted CD4+ T cells were non-specifically maintained by culturing in RPMI 1640 media supplemented with penicillin/streptomycin, 10% fetal bovine albumin and 100 ng/ml Concanavalin A (Sigma- Aldrich; Saint Louis, MO, USA) during the generation of MoDCs from sorted monocytes. Monocytes were cultured at a concentration of 0.75 - 1 x 106/ml for 5 days in RPMI supplemented with penicillin/streptomycin, 10% FBS, 100 ng/ml
  • Granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng IL-4 (R&D Systems, Minneapolis, MN, USA).
  • GM-CSF Granulocyte-macrophage colony-stimulating factor
  • IL-4 R&D Systems, Minneapolis, MN, USA
  • MoDCs were harvested and incubated in the absence or presence of 5 ⁇ g/ml CRM197 (Sigma-Aldrich, Saint Louis, MO, USA) for 5 hours in RPMI without FBS. After 5 hours, FBS was added to 10% (v/v) and 100 ng/ml lipopolysaccharide (ultra-pure from List Biological Laboratories; Campbell, CA, USA) was added for an additional 18 hours.
  • MoDCs were subsequently harvested, washed and co-cultured at 5,000 cells per well with 50,000 T cells for 7 days.
  • IL-4 or IL-17 were analyzed by intracellular cytokine staining after the addition of BD Golgi-plug (BD Biosciences) during the final 6 hours of culture. T cells were made permeable with Cytofix/Cytoperm reagents (BD Biosciences). Cells were stained with anti-IFN ⁇ -PE.Cy7 (clone B27, BD Biosciences), anti-IL- 17-APC (clone 41802, R&D Systems), and anti-IL-4-V450 (clone 8D4-8, BD Biosciences).
  • ELISAs Quantitation of total S. pneumoniae and capsular polysaccharides serotype 4-, 6B-, 14-, and 23F-specific IgG were determined by use of an adapted WHO recommended ELISA protocol, as outlined by the Bacterial Respiratory Pathogen Reference Laboratory at the University of Alabama at Birmingham (on the wold wide web at
  • Serum Ab concentrations were calculated by comparing the optical density of each unknown well at 405 nm and 690 nm (reference), and to the optical density of the standard (human anti-pneumococcal reference serum, lot 89-SF). For avidity determination, assessment of the overall strength of binding between Ab and antigen, a 0 - 4 M NaSCN gradient was used to determine the NaSCN concentration that competes off approximately 50% of the bound rhesus immunoglobulins (56).
  • Results were considered significant at p ⁇ 0.05, and indicated as follows: +p ⁇ 0.05, ++p ⁇ 0.01, +++p ⁇ 0.001, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Neonatal pneumococcal conjugate vaccine immunization primes T cells for preferential Th2 cytokine expression: a randomized controlled trial in Papua New Guinea. Vaccine. 2009;27(9): 1340-7.
  • TIR Toll/Interleukin-1 Receptor
  • Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies. J Immunol. 2006; 176(9):5671-81.
  • Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Thl immunity with poly IC as adjuvant. J Exp Med.
  • HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Thl and CD8+ T cell responses in nonhuman primates. Proc Natl Acad Sci U S A. 2005; 102(42): 15190-4.
  • hyporesponsiveness to neonatal acellular pertussis vaccination in a mouse model hyporesponsiveness to neonatal acellular pertussis vaccination in a mouse model.
  • Infection is the most common cause of mortality early in life, in large measure due to suboptimal early life vaccination strategies as compared to older age groups.
  • New adjuvants are absolutely cardinal to further optimize current immunization approaches.
  • only a few classes of adjuvants are presently incorporated in vaccines approved for human use.
  • TLR7/8 adjuvant strategies described herein may be key to enhance early life immunogenicity by creating a vaccine formulation that induces both robust and persistent immunity (i.e., overcome waning immunity) to B. pertussis. Described herein is the optimization of a) the formulation delivery system, b) stability, and c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes.
  • TLR7/8 adjuvant can overcome neonatal hyporesponsiveness to acellular pertussis vaccination by driving Thl favoring responses to a licensed acellular vaccine (DTaP).
  • This potent immunization strategy is of fundamental importance in vaccine development and represents a new paradigm for effective pertussis immunization in early life.
  • Infection is the most common cause of mortality early in life, substantively due to suboptimal vaccination strategies for newborns and infants as compared to older age groups.
  • the development of an effective infant pertussis vaccine has become urgent because of the resurgence of pertussis in many countries.
  • Current alum adjuvanted acellular pertussis (aP) vaccines have various shortcomings, which may contribute to their suboptimal infant responses.
  • New adjuvants may allow for the development of new vaccines and/or further optimizing current immunization approaches.
  • age-focused adjuvant strategies may be the most singular solution to enhance early life immunogenicity by creating a vaccine formulation that induces both robust and persistent immunity to Bordetella pertussis.
  • Described herein is the selection of a core TLR7/8 compound structure based on it's strong in vitro activity towards human newborn leukocytes, modification of it by lipidation and absorption onto alum.
  • the alum adsorbed TLR7/8 adjuvant enhanced antibody responses to a licensed acellular vaccine, by polarizing towards Thl favoring responses.
  • This strategy represents a new example of how a combined alum + TLR7/8 adjuvant approach could reshape our current immunization schedules with significant potential to impact B.
  • Alum based subunit vaccines consisting of purified microbial products often lack the necessary adjuvant activity to induce and optimally shape an immune response [10]. Accordingly, development of rationally designed vaccine formulations, which include adjuvants that more effectively enhance immune responses in childhood, are warranted [11, 12]. In this context, TLR7/8 agonists are
  • IMQs TLR7/8 ligands do not work well in comparison to their larger molecular weight counterparts' such as alum, since they are prone to diffuse away from the injection site, which can also result in systemic toxicity [21].
  • R848 has a poor tolerability profile when tested systemically in humans.
  • Common systemic side effects of this aqueous formulation include injection site reactogenicity and flu-like symptoms (fever, headache and malaise) correlating to systemic immune activation, seen with high concentrations of numerous inflammatory cytokines in the blood [22, 23].
  • TLR7/8 adjuvantation can overcome newborn
  • 3M-052 a locally- acting lipidated FMQ TLR7/8 agonist adjuvant bearing a fatty acid tail (CI 8 lipid moiety), was shown to drive robust T helper 1 -cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine (PCV)13 [24]. Moreover, a single administration of 3M-052 in combination with PCV13 on the first day of life, accelerated and enhanced anti-S.
  • PCV pneumococcal conjugate vaccine
  • TLR7/8 agonist CRX-649 demonstrates robust newborn Thl polarizing immune activity.
  • IMQ novel imidaziquinoline
  • OA oxoadinine
  • CRX-672 and three OA compounds, CRX-672, CRX-677 and CRX-748 (Fig. Fig. 25A, 25C) were found to have variable TLR7 and TLR8 selectivity and potency in HEK293 assay (Fig. 25 A, and Figs. 26A- 26C). Though their TLR7 vs. TLR8 potency or selectivity did not correlate to their structure or side chain modifications, CRX-649 had the greatest potency for both hTLR7 and hTLR8 (Figs. 26A-26C).
  • Figs. 27A-27D the ability of the core IMQs and OAs to induce concentration-dependent leukocyte cytokine production in age-specific in vitro human neonatal and adult blood models was tested.
  • All six of the TLR agonists tested demonstrated titration-dependent induction of TNF (Fig. 27A) and IFNy (Fig. 27B) from the neonatal cells.
  • the vehicle control did not induce TNF or IFNy production.
  • CRX-649 demonstrated a more than adultlike age-specific potency and effectiveness for TNF production in newborn cord blood (Fig. 27C).
  • CRX-649 demonstrated the greatest potency, effectiveness and IFNy polarization in newborn cord blood.
  • CRX-649 mediated IFNy production in newborn blood was most evident at 10 ⁇ , reaching -1200 pg/ml, twice the produced in similarly treated adult blood, (Fig. 27D, p ⁇ 0.0 ⁇ ).
  • CRX-649 also demonstrated a broader ability to induce a newborn-specific cytokine and chemokine potency and polarization.
  • whole blood treated supernatants were analyzed for cytokine/chemokine/interferon expression by multiplex assay, and the results graphed as fold change for newborn cold over adult, at both a low (1 ⁇ , Figs. 28 A, 28B) and high (10 ⁇ , Figs. 28C, 28D) concentration of CRX-649, induced concentration-dependent production of IL- 1 ⁇ , IL-6, IL-10, and IL-12p40 (Figs. 28A, 28C).
  • CXCL8, CXCL10, CCL2 and GM-CSF also demonstrated greater CRX-649 induced production in newborn blood (Figs. 28B, 28D).
  • a human newborn Thl polarization assay was employed, which leverages the intrinsic characteristics of the newborn T cell compartment (composed mainly of naive T cells) to evaluate how CRX-649 modulated T cell polarization in a mixed mononuclear cell culture in the presence of a TCR- mediated stimulus.
  • CBMCs were cultured in the presence of autologous plasma, a rich source of age-specific soluble immunomodulatory factors [25].
  • this core was further derivitized by addition of a phosphate off the ethanol at the 1 position, followed by addition of an optimally determined 3 PEG linker (Fig. 29C).
  • the core compound CRX-649 was compared for cytokine induction from adult PBMCs in relation to the phosphorylated derivative, CRX-650, or the phospholipidated derivative, CRX- 727. While the core compound does not contain a phosphate moiety and thus cannot adsorb to alum, present in many vaccines as adjuvants and antigen stabilizers, the addition of the phosphate on the core can facilitate this.
  • the nucleolipid derivative compound(s) contain phosphate groups which are theoretically also able to adsorb to alum in the vaccine. Thus an adsorption study was undertaken to determine how the different FMQ adjuvant compounds would interact with the final vaccine antigen formulation (i.e., DTaP).
  • DTaP final vaccine antigen formulation
  • Aqueous suspensions of the lipidated compound CRX-727 and its parent pharmacophore CRX-649 were incubated with DTaP vaccine and aliquots were assayed at various time points (1, 2, 24 hrs) post admixture to assess amounts of unbound compounds in the supernatant via reversed-phase high-performance liquid
  • lipidated compound fully adsorbed (-96 - 100 %) to the alum/antigen within 1 hr (Fig. 30, top).
  • the core CRX-649 compound was only able to adsorb to the antigen ⁇ 4 - 7 % within 1-2 hr (Fig. 30, bottom panels), with peak area intensity levels similar to the unmixed controls.
  • mice were immunized twice, 14 days apart with Infanrix (1/lOOth of the human dose) ⁇ CRX-649 or CRX-727 at 0.1, 1 or 10 ⁇ g per mouse in different formulations. Serum was harvested 14 days following prime and boost (Fig. 31 A). At two weeks post primary vaccination, anti-FHA IgG2a serum antibody titers were significantly elevated over antigen alone vaccinated mice when 10 ⁇ g of CRX-727, with (p ⁇ 0.0001) or without alum (p ⁇ 0.001) pre-adsorption of the adjuvant, was included in the vaccine (Fig.
  • IgGl anti-FHA titers were only significantly boosted vs. antigen alone with CRX-727 pre-adsorbed on alum (p ⁇ 0.001) (Fig. 31 A, left) demonstrating a potential difference in effect for alum adsorption in driving a Thl vs. Th2 skewed immune response.
  • CRX-727 + alum also induced significantly enhanced IgG2a as compared to CRX-727 post primary vaccination (Fig. 31 A).
  • imidaziquinoline CRX-649 was not able to enhance either IgGl or IgG2a antibody titers in the adult mice.
  • potency increases were observed when boosting with a 1 ⁇ g dose CRX-727 alone (p ⁇ 0.0001) and a 0.1 ⁇ g dose CRX-727 + alum (Fig. 33C, p ⁇ 0.01).
  • Fig. 33C, p ⁇ 0.01 show that it is either the adsorption of the adjuvant to the alum or type I interferon skewing that is necessary for enhancement of immune response to this vaccine. This is further supported from data examining the cell-mediated immune response of vaccinated animals.
  • TLR7/8 adjuvant formulated with alum overcomes neonatal hyporesponsisves to DTaP. Having demonstrated the potential of alum adsorbed lipidated TLR7/8 adjuvanting to enhance both the correlates of immunogenicity and antibody subclass induction in adult mice, it was assessed if the same phenotype is achievable in a neonatal setting. Of note, vaccine driven antibody isotype switching toward IgG2c, with alum as the sole adjuvant, is diminished or not achievable in early life [31].
  • Rational vaccine design approaches employing immunoengineering and novel delivery systems may allow for the controlled preparation of vaccine complexes of the desired immuno-stimulatory properties, particulate size, and antigen load, all of which also improve safety by potentially limiting systemic toxicities by their targeted nature [33].
  • the persistently high global burden of infections in the very young [34] provides a compelling rationale for developing additional safe and effective early life vaccines.
  • the present project synergistically combined three innovative approaches to provide unique insight into and overcome early life aP vaccine hyporesponsiveness: 1) the use of age specific human in vitro and murine in vivo models, 2) the employment of cutting edge medicinal chemistry and 3) formulation science techniques to optimize small molecule adjuvanticity and delivery.
  • Next generation pertussis vaccines might require increased amounts of appropriate antigens and potentially novel adjuvantation systems as well.
  • relatively low reactogenicity will be an important feature of successful next generation pertussis vaccines.
  • mice were housed in couples, and cages checked daily to assess pregnancy status of dams and/or the presence of pups.
  • Human blood was anti-coagulated with 20 units/ml pyrogen-free sodium heparin (American Pharmaceutical Partners, Inc.; Schaumberg, IL). All blood products were kept at room temperature and processed within 4 hours from collection. Human whole blood assays were completed as previously described [14, 17].
  • PBMCs For adult PBMC stimulation only, primary human PBMCs were isolated from fresh blood from healthy donors via Ficoll gradient separation. PBMCs were resuspended and maintained in RPMI- 1640 culture media (Invitrogen, Grand Island, NY), antibiotics
  • cytokine and chemokine expression profiles in cell culture supernatants and peripheral blood plasma were measured using customized MilliplexTM human cytokine/chemokine magnetic bead panels (Millipore; Billerica, MA, USA). Assays were analyzed on the Luminex® 100/200TM System employing xPOTENT® software (Luminex; Austin, TX) and Millipore Milliplex AnalystTM (version 3.5.5.0). The minimum threshold for each analyte was set at the minimum detectable concentration for a given assay, defined as three standard deviations above the mean background. CL075 (TLR8/7) was purchased from InvivoGen (San Diego, CA) and used at the concentrations noted in the figure legends.
  • CRX-727 adsorption to aluminum hydroxide 100 ⁇ l of CRX-727 was mixed with 100 ⁇ l of InfanrixTM (combination vaccine for diphtheria, tetanus, and acellular pertussis (DTaP)) (a 1 : 10 CRX-727:alum mass ratio) plus 300 ⁇ l of 0.9% saline. After vortexing for 10 seconds the sample was placed in a 37°C incubator. Every 15 minutes the sample was vortexed for an additional 5 seconds and placed back into the incubator.
  • InfanrixTM combination vaccine for diphtheria, tetanus, and acellular pertussis (DTaP)
  • a gradient was performed using a two mobile phase system of 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, on an Agilent Zorbax Eclipse Plus CI 8, 4.6 x 150 mm, 5 micron column at 25 °C.
  • the response (peak area) of the samples were compared against a 50 ⁇ l CRX-727 plus 200 ⁇ l 0.9% saline control and a separate 100 ⁇ l alum plus 400 ⁇ 1 saline control.
  • CBMCs Newborn cord blood mononuclear cells
  • TLR7/8 agonists in the presence of the polyclonal T cell activator aCD3 for 96 hours.
  • T cell polarization was evaluated by IFNy levels measured in cell-free supernatants by ELISA.
  • mice In vivo rodent immunization, antigens, and antibody quantification.
  • both neonate and adult mice were immunized intramuscularly (i.m.) in the posterior thigh with 50 ⁇ l of total vaccine dose.
  • Balb/c mice (6-8 weeks of age) were immunized with Infanrix (GSK, 1/100th of the human dose) ⁇ CRX649 or CRX-727 at 0.1 ⁇ g, 1 ⁇ g or 10 ⁇ g per mouse in different formulations (aqueous choline salt, liposome or alum pre-adsorbed).
  • prime-boost schedule two injections one week apart, for newborn mice at DOL 7 and 14.
  • FHA hemagglutinin
  • Cells were then washed, stained for viability (Tonbo Biosciences ghost 510) and then surface stained for 30 min with anti-CD3, CD 8 and CD4 antibodies (clones 145-2C11, 25-0042, 60-0081 (all Tonbo), respectively). Cells were then washed, fixed/permeabilized (BD fix/per buffer, BD Biosciences) and intracellularly stained for 30 min with and IFNy (XMG1.2 (BD) antibody. Cells were then washed and analyzed on an LSR II flow cytometer (BD) using FACSDivaTM software and FlowJoTM vlO software for post-acquisition analysis.
  • BD LSR II flow cytometer
  • HEK293 assay for human TLR7 and TLR8 selectivity Human embryonic kidney (HEK)293 cells expressing human TLR7 or TLR8 with an NF-KB-responsive secreted embryonic alkaline phosphatase (SEAP) reporter gene were obtained from Novus Biologicals (Littleton, CO) and Invivogen (San Diego, CA), respectively. Cells were maintained in DMEM with 10% HI-FBS and selection antibiotics per the manufacturer's instructions. Cells were plated at 5x l0 5 cells/96-well and stimulated with indicated agonist(s) for 24 h. Supernatants were harvested and analyzed for NF-KB/ SEAP activation using the QuantiBlueTM kit (Invivogen). Values are expressed as fold change in OD 6 5o over vehicle-only treated samples.
  • SEAP embryonic alkaline phosphatase
  • PubMed PMID 27081760; PubMed Central PMCID: PMCPmc4906944. 11. Dowling DJ, Levy O. Pediatric Vaccine Adjuvants: Components of the Modern
  • Imidazoquinoline Toll-like receptor 8 agonists activate human newborn monocytes and dendritic cells through adenosine-refractory and caspase-1 -dependent pathways. J Allergy Clin Immunol. 2012; 130(1): 195-204 e9. Epub 2012/04/24. doi: 10.1016/j .jaci.2012.02.042. PubMed PMID: 22521247; PubMed Central PMCID: PMC3387351.

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Abstract

Les procédés et les compositions de l'invention concernent des procédés d'immunisation ou de stimulation d'une réponse immunitaire, par exemple, à l'aide d'agonistes de TLR7 et/ou TLR8 en tant qu'antigènes. Les procédés et les compositions de l'invention ont une pertinence particulière pour une utilisation chez les nourrissons.
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